Angiogenically effective unit dose of FGF and method of administering

ABSTRACT

The present invention has multiple aspects. In particular, in one aspect, the present invention is directed to a unit dose comprising 0.2 μg/kg to 36 μg/kg of a recombinant FGF or an angiogenically active fragment or mutein thereof. In another aspect, the present invention is directed to a pharmaceutical composition comprising an angiogenically effective dose of an FGF or an angiogenically active fragment or mutein thereof, and a pharmaceutically acceptable carrier. Typically, the angiogenically effective dose comprises 0.2 μg/kg to 36 μg/kg of an FGF of any one of SEQ ID NOS: 1-3, 5, 8-10, or 12-14 or an angiogenically active fragment or mutein thereof. In yet another aspect, the present invention is directed to a method for treating a human patient for coronary artery disease, comprising administering into at least one coronary vessel of a human patient in need of treatment for coronary artery disease a safe and angiogenically effective dose of a recombinant FGF of any one of SEQ ID NOS: 1-3, 5, 8-10, or 12-14, or an angiogenically active fragment or mutein thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation of U.S. application Ser. No.09/417,721, filed Oct. 13, 1999, which claims the benefit of U.S.Application Serial No. 60/104,103, filed Oct. 13, 1998, whichapplications are herein incorporated by reference in their entirety.

[0002] 1. Field of the Invention

[0003] The present invention is directed to a unit dose comprising anFGF or an angiogenically active fragment or mutein thereof for inducingcardiac angiogenesis in a human. The present invention is also directedto a pharmaceutical composition; comprising the unit dose of the FGF andto a method for administering this pharmaceutical composition to a humanto induce cardiac angiogenesis while minimizing systemic risk to thepatient. The present invention is useful because the unit dose,pharmaceutical composition and the method for its administration providean alternative to surgical intervention for the treatment of coronaryartery disease (CAD) and may fuirther provide an adjunct for reducingpost myocardial infarct (MI) injury in humans.

[0004] 2. Background of the Invention

[0005] The fibroblast growth factors (FGF) are a family of at leasteighteen structurally related polypeptides (named FGF-1 to FGF-18) thatare characterized by a high degree of affinity for proteoglycans, suchas heparin. The various FGF molecules range in size from 15-23 kD, andexhibit a broad range of biological activities in normal and malignantconditions including nerve cell adhesion and differentiation [Schubertet al., J. Cell Biol. 104:635-643 (1987)]; wound healing [U.S. Pat. No.5,439,818 (Fiddes)]; as mitogens toward many mesodermal and ectodermalcell types, as trophic factors, as differentiation inducing orinhibiting factors [Clements, et al., Oncogene 8:1311-1316(1993)]; andas an angiogenic factor [Harada, J. Clin. Invest., 94:623-630 (1994)].Thus, the FGF family is a family of pluripotent growth factors thatstimulate to varying extents fibroblasts, smooth muscle cells,epithelial cells, and neuronal cells.

[0006] When FGF is released by normal tissues, such as in fetaldevelopment or wound healing, it is subject to temporal and spatialcontrols. However, many of the members of the FGF family are alsooncogenes. Thus, in the absence of temporal and spatial controls, theyhave the potential to stimulate tumor growth by providing angiogenesis.

[0007] Coronary artery disease (atherosclerosis) is a progressivedisease in humans wherein one or more coronary arteries gradually becomeoccluded through the buildup of plaque. The coronary arteries ofpatients having this disease are often treated by balloon angioplasty orthe insertion of stents to prop open the partially occluded arteries.Ultimately, these patients are required to undergo coronary arterybypass surgery at great expense and risk. It would be desirable toprovide such patients with a medicament that would enhance coronaryblood flow so as to preclude the need to undergo bypass surgery.

[0008] An even more critical situation arises in humans when a patientsuffers a myocardial infarction, wherein one or more coronary arteriesor arterioles becomes completely occluded, such as by a clot. There isan immediate need to regain circulation to the portion of the myocardiumserved by the occluded artery or arteriole. If the lost coronarycirculation is restored within hours of the onset of the infarction,much of the damage to the myocardium that is downstream from theocclusion can be prevented. The clot-dissolving drugs, such as tissueplasminogen activator (tPA), streptokinase, and urokinase, have beenproven to be useful in this instance. However, as an adjunct to the clotdissolving drugs, it would also be desirable to also obtain collateralcirculation to the damaged or occluded myocardium by angiogenesis.

[0009] Accordingly, it is an object of the present invention to providea medicament and a mode of administration that provides human patientswith cardiac angiogenesis during coronary artery disease and/or postacute myocardial infarction. More particularly, it is a further objectof the present invention to provide a therapeutic dose of an FGF and amode of administration to humans that provide the desired property ofcardiac angiogenesis, while minimizing adverse effects.

[0010] Many of the various FGF molecules have been isolated andadministered to various animal models of myocardial ischemia withvarying and often times opposite results. According to Battler et al.,“the canine model of myocardial ischemia has been criticized because ofthe abundance of naturally occurring collateral circulation, as opposedto the porcine model, which ‘excels’ in its relative paucity of naturalcollateral circulation and its resemblance to the human coronarycirculation.” Battler et al., “Intracoronary Injection ofBasicFibroblast Growth Factor Enhances Angiogenesis in Infarcted SwineMyocardium, ” JACC, 22(7):2001-6(Dec. 1993) at page 2002, col. 1.However, Battler et al., who administered bovine bFGF (i.e., FGF-2) topigs in a myocardial infarct model, considered the varying results thatare obtained from one animal species to another, and expressly disclosesthat the divergent results “thus emphasiz[e] the caution that must beexercised in extrapolating results from different animal models.”Battler et al., at page 2005, col.1. Further, Battler points out that“the dosage and mode of administration of bFGF [i.e., bovine FGF-2] mayhave profound implications for the biologic effect achieved.” Battler,et al., at page 2005, col. 1. Thus, it is a further object of thisinvention to discover a dosage and a mode of administration of afibroblast growth factor that would provide for the safe and efficacioustreatment of CAD and/or post MI injury in a human patient. Moregenerally, it is an object of the present invention to provide apharmaceutical composition and method for inducing angiogenesis in ahuman heart.

SUMMARY OF THE INVENTION

[0011] The Applicants have discovered that a fibroblast growth factor,such as of SEQ ID NOS: 1-3, 5, 8-9, or 12-14 or an angiogenically activefragment or mutein thereof, when administered as a unit dose of about0.2 μg/kg to about 36 μg/kg into one or more coronary vessels (IC) of ahuman patient in need of coronary angiogenesis, unexpectedly providesthe human patient with a rapid and therapeutic cardiac angiogenesissufficient to obviate surgical intervention and results in anunexpectedly superior increase in the treated patient's exercisetolerance time (ETT). By way of comparison, angioplasty is considered atherapeutic success if it provides an increase in a patient's ETT ofgreater than 30 seconds compared to the placebo. By the term “cardiacangiogenesis” or “coronary angiogenesis,” as used herein, is meant theformation of new blood vessels, ranging in size from capillaries toarterioles which act as collaterals in coronary circulation.

[0012] FGFs that are suitable for use in the present invention includehuman FGF-1 (SEQ ID NO: 1), bovine FGF-1 (SEQ ID NO: 2), human FGF-2(SEQ ID NO: 3), bovine FGF-2(SEQ ID NO: 5), human FGF-4(SEQ ID NO: 8),human FGF-5(SEQ ID NO: 9), human FGF-6(SEQ ID NO: 10), human FGF-8(SEQID NO: 12), human FGF-9(SEQ ID NO: 13), and human FGF-98(SEQ ID NO: 14).In one embodiment, FGF molecules are human FGF-1(SEQ ID NO: 1), bovineFGF-1(SEQ ID NO: 2), human FGF-2(SEQ ID NO: 3), bovine FGF-2(SEQ ID NO:5), human FGF-4(SEQ ID NO: 8), and human FGF-5(SEQ ID NO: 9). In analternative embodiment, the FGF molecules are human FGF-6(SEQ ID NO:10), murine FGF-8 (SEQ ID NO: 12), human FGF-9(SEQ ID NO: 13) or humanFGF-98(SEQ ID NO: 14).

[0013] Typically, the angiogenically active fragments of the presentinvention retain the distal two thirds of the mature FGF molecule (i.e.,the two thirds of the molecule at the carboxy end that have the cellbinding sites). For convenience, the terms “human FGF,” “bovine FGF,”and murine FGF are used herein in abbreviated form as “hFGF,” “bFGF,”and “mFGF,” respectively.

[0014] The Applicants also discovered that a single unit dose of an FGFor an angiogenically active fragment thereof, when administered as aunit dose into one or more coronary vessels (IC) of a human patient inneed of coronary angiogenesis (e.g., a human patient with coronaryartery disease despite optional medical management), unexpectedlyprovides the human patient with a therapeutic benefit that is seen asearly as two weeks after the single unit dose is administered (asreflected in symptoms), and that lasts at least 60 days after the singleunit dose is administered (as reflected in ETT and the “Seattle AnginaQuestionnaire” (SAQ)). For example, when 28 human patients diagnosed ashaving CAD were assessed by the SAQ both before and 57 days after beingadministered IC a single unit dose of 0.33 μg/kg to 48 μg/kg of FGF-2 ofSEQ ID NO: 5, the mean increase in their scores on the five criteriaassessed ranged from 13 to 36 points, which is about 1.6-4.5 timesgreater than the 8 point change which was considered to be “clinicallysignificant” in alternative modes of treatment. See Table 2. When thescores of the 15 first patients were broken down between those receivinga low dose (less than 2 μg/kg) and those receiving a higher dose(greater than or equal to 2 μg/kg) of FGF-2 of SEQ ID NO: 5, andassessed by the SAQ, both doses were found to provide scores that had“clinically significant” increases ranging from 12.3 to 58.1 and 10.9 to32.1, respectively. Thus, whether the patients were administered thelower doses or the higher doses of the invention, their increased scoreswere about 1.4-7.2 times greater than the 8 point change which wasconsidered to be “clinically significant” in alternative modes oftreatment. See Table 3.

[0015] As part of this study, MRI was performed on 23 human patientsdiagnosed with CAD to assess ejection fraction, regional myocardialfunction and perfusion (delayed arrival zone). The patients wereadministered IC a single unit dose of 0.33 μg/kg to 12 μg/kg of FGF-2 ofSEQ ID NO: 5. Their cardiac and coronary functions were objectivelyassessed by magnetic resonance imaging (MRI) both before and aftertreatment. The MRI results demonstrated significant improvement inregional wall motion (%) and wall thickening (%) during systole. Theresults also showed a significant reduction in the delayed arrival zone(% LV). The results did not demonstrate any significant change inejection fraction (EF). Thus, the Applicants have demonstrated theclinical efficacy in humans of a single unit dose of an FGF whenadministered IC in accordance with the present invention.

[0016] Accordingly, in one aspect, the Applicants' invention is directedto a unit dose of FGF comprising a safe and therapeutically effectiveamount of an FGF of any one of SEQ ID NOS: 1-3, 5, 8-10, or 12-14 or anangiogenically active fragment or mutein thereof. Typically, the safeand therapeutically effective amount comprises about 0.2 μg/kg to about36 μg/kg of an FGF of any one of SEQ ID NOS: 1-3, 5, 8-10, or 12-14 oran angiogenically active fragment or mutein thereof. In otherembodiments, the safe and therapeutically effective amount of the unitdose comprises 0.2 μg/kg to 2.0 μg/kg, 2.0 μg/kg to 20 μg/kg, or 20μg/kg to 36 μg/kg of an FGF of any one of SEQ ID NOS: 1-3, 5, 8-10, or12-14 or an angiogenically active fragment or mutein thereof. Expressedin absolute terms for the majority of human CAD patients, the unit doseof the present invention comprises .008 mg to 6.1 mg, more typically 0.3mg to 3.5 mg, of the FGF of any one of SEQ ID NOS: 1-3, 5, 8-10, or12-14 or an angiogenically active fragment or mutein thereof.

[0017] In another aspect, the present invention is directed to apharmaceutical composition comprising a safe and therapeuticallyeffective amount of an FGF or an angiogenically active fragment ormutein thereof, and a pharmaceutically acceptable carrier. Typically,the safe and therapeutically effective amount of an FGF comprises about0.2 μg/kg to about 36 μg/kg of an FGF of any one of SEQ ID NOS: 1-3, 5,8-10, or 12-14 or an angiogenically active fragment or mutein thereof,and a pharmaceutically acceptable carrier. In other embodiments of thepharmaceutical composition, the safe and therapeutically effectiveamount of an FGF comprises 0.2 μg/kg to 2 μg/kg, 2 μg/kg to 20 μg/kg, or20 μg/kg to 36 μg/kg of an FGF, such as an FGF of any one of SEQ ID NOS:1-3, 5, 8-10, or 12-14 or an angiogenically active fragment or muteinthereof, and a pharmaceutically acceptable carrier.

[0018] In yet another aspect, the present invention is directed to amethod of using the above described unit dose or pharmaceuticalcomposition to treat a human patient for CAD or to induce coronaryangiogenesis therein. The method comprises administering into one ormore coronary vessels of a human patient in need of treatment forcoronary artery disease (or in need of angiogenesis) a safe andtherapeutically effective amount of a recombinant FGF or anangiogenically active fragment or mutein thereof. Typically, a portionof the safe and therapeutically effective amount is administered to eachof two coronary vessels. More typically, the safe and therapeuticallyeffective amount comprises about 0.2 μg/kg to about 36 μg/kg of an FGFof any one of SEQ ID NOS: 1-3, 5, 8-10, or 12-14 or an angiogenicallyactive fragment or mutein thereof in a pharmaceutically acceptablecarrier. In other embodiments, the safe and therapeutically effectiveamount comprises 0.2 μg/kg to 2 μg/kg, 2 μg/kg to 20 μg/kg, or 20 μg/kgto 36 μg/kg of the FGF of any one of SEQ ID NOS: 1-3, 5, 8-10, or 12-14or an angiogenically active fragment or mutein thereof in apharmaceutically acceptable carrier.

[0019] Because FGF is a glycosoaminoglycan (e.g., heparin) bindingprotein and the presence of a glycosoaminoglycan (also known as a“proteoglycan” or a “mucopolysaccharide”) optimizes activity and AUC,the IC dosages of the FGF of the present invention typically areadministered within 20 minutes of the IV administration of aglycosoaminoglycan, such as a heparin.

BRIEF DESCRIPTION OF THE FIGURES

[0020]FIG. 1 is a plot of the mean rFGF-2 plasma concentration versustime (hours) for six different doses of rFGF-2 (SEQ ID NO: 5)administered by IC infusion in humans over a 20 minute period. The sixdoses of rFGF-2 in FIG. 1 are 0.33 μg/kg, 0.65 μg/kg, 2 μg/kg, 6 μg/kg,12 μg/kg, and 24 μg/kg of lean body mass (LBM).

[0021]FIG. 2 is a plot of each individual patient's rFGF-2 area underthe curve (AUC) in pg·hr/ml for FIG. 1 for the six doses of rFGF-2, andshows the dose linearity of systemic rFGF-2 exposure following ICinfusion.

[0022]FIG. 3 is a plot individual human patient rFGF-2 dose normalizedAUCs as a function of the time of heparin administration in “minutesprior to rFGF-2 infusion” and shows the influence of timing of heparinadministration on rFGF-2 AUC.

DETAILED DESCRIPTION OF THE INVENTION

[0023] The Applicants have discovered that single dose of an FGF or anangiogenically active fragment or mutein thereof, when administered in asafe and therapeutically effective amount into one or more coronaryvessels of a human patient diagnosed with CAD provides the patient witha safe and therapeutically efficacious treatment for the patient'scoronary artery disease that lasts at least 6 months before a furthertreatment is needed. In fact, the Applicants' method for treating CAD,when assessed by the standard objective criterion employed in the art(i.e., ETT), provided an unexpectedly superior increase of one and ahalf to two minutes in the treated patient's ETT. This comparesexceptionally well when compared to the increase of 30 seconds that isdeemed clinically significant for the current mode of treatment, i.e.,angioplasty.

[0024] By the phrase “safe and therapeutically effective amount” as usedherein in relation to FGF is meant an amount of an FGF or anangiogenically active fragment or mutein thereof that when administeredin accordance with this invention, is free from major complications thatcannot be medically managed, and that provides for objective cardiacimprovement in patients having symptoms of CAD despite optimum medicalmanagement. Thus, acute hypotension that can be managed byadministration of fluids, with no other side effects is considered“safe” for the purpose of this invention. Typically, the safe andtherapeutically effective amount of an FGF comprises about 0.2 μg/kg toabout 36 μg/kg of the FGF of any one of SEQ ID NOS: 1-3, 5, 8-10, or12-14 or an angiogenically active fragment or mutein thereof.

[0025] Accordingly, the present invention has multiple aspects. In itsfirst aspect, the present invention is directed to a unit dose of theFGF medicament that has produced unexpectedly superior results intreating CAD in humans when compared to angioplasty. In particular, theunit dose comprises a safe and therapeutically effective amount of anFGF or an angiogenically active fragment or mutein thereof. Typically,the unit dose comprises about 0.2 μg/kg to about 36 μg/kg of the FGF ofany one of SEQ ID NOS: 1-3, 5, 8-10, or 12-14 or an angiogenicallyactive fragment or mutein thereof. In other embodiments of the unitdose, the safe and therapeutically effective amount comprises about 0.2μg/kg to about 2 μg/kg, about 2 μg/kg to about 20 μg/kg, or about 20μg/kg to about 36 μg/kg of the FGF of any one of SEQ ID NOS: 1-3, 5,8-10, or 12-14 or an angiogenically active fragment or mutein thereof.It is convenient to provide the unit dose of the present invention in aformulation comprising in absolute terms from .008 mg to 6.1 mg of theFGF of any one of SEQ ID NOS: 1-3, 5, 8-10, or 12-14 or anangiogenically active fragment or mutein thereof. In this embodiment,the unit dose contains a sufficient amount of FGF to accommodate dosingany one of the majority of CAD patients, ranging from the smallestpatient (e.g., 40 kg) at the lowest dosage (about 0.2 μ/kg) through thelarger patients (e.g., 170 kg) at about the highest dosage (about 36μg/kg). More typically, the unit dose comprises 0.3 mg to 3.5 mg of theFGF of any one of SEQ ID NOS: 1-3, 5, 8-10, or 12-14 or anangiogenically active fragment or mutein thereof. The unit dose istypically provided in solution or lyophilized form containing the abovereferenced amount of FGF and an effective amount of one or morepharmaceutically acceptable buffers, stabilizers and/or other excipientsas later described herein.

[0026] The active agent in the above described unit dose is arecombinant FGF or an angiogenically active fragment or mutein thereof.Typically, the active agent of the unit dose is the FGF of any one ofSEQ ID NOS: 1-3, 5, 8-10, or 12-14. More typically, the active agent inthe unit dose is hFGF-1(SEQ ID NO: 1), bFGF-1(SEQ ID NO: 2), hFGF-2(SEQID NO: 3), bFGF-2(SEQ ID NO: 5), hFGF-4(SEQ ID NO: 8) or hFGF-5(SEQ IDNO: 9). In an alternative embodiment, the active agent in the unit doseis hFGF-6(SEQ ID NO: 10), mFGF-8(SEQ ID NO: 12), hFGF-9(SEQ ID NO: 13)or hFGF-98(SEQ ID NO: 14).

[0027] The amino acid sequences and methods for making many of themammalian FGFs that are employed in the unit dose, pharmaceuticalcomposition and method of the present invention are well known in theart. In particular, references disclosing the amino acid sequence andrecombinant expression of FGF 1-9 and FGF-98 are discussed sequentiallybelow.

[0028] FGF-1: The amino acid sequence of HFGF-1(SEQ ID NO: 1) and itsrecombinant expression are disclosed in U.S. Pat. No. 5,604,293(Fiddes), entitled “Recombinant Human Basic Fibroblast Growth Factor,”which issued on Feb. 18, 1997. See FIG. 2d of the '293 patent. Thisreference and all other references herein, whether cited before or afterthis sentence, are expressly incorporated herein by reference in theirentirety. The amino acid sequence and recombinant expression ofbFGF-1(SEQ ID NO: 2) are also disclosed in U.S. Pat. No. 5,604,293(Fiddes) which has been incorporated herein by reference. See, FIG. 1bof the '293 patent. Both HFGF-1(SEQ ID NO: 1) and bFGF-1(SEQ ID NO: 2)have 140 amino acid residues. bFGF-1differs from hFGF-1at 19 residuepositions: 5(Pro→Leu), 21(His→Tyr), 31(Tyr→Val), 35(Arg→Lys),40(Gln→Gly), 45(Gln→Phe), 47(Ser→Cys), 51(Tyr→Ile), 54(Tyr→Val),64(Tyr→Phe), 80(Asn→Asp), 106(Asn→His), 109(Tyr→Val), 116(Ser→Arg),117(Cys→Ser), 119(Arg→Leu), 120(Gly→Glu), 125(Tyr→Phe), and137(Tyr→Val). In most instances, the differences are conserved. Further,the differences at residue positions 116 and 119 merely interchange theposition of the Arg.

[0029] FGF-2: The amino acid sequence of hFGF-2(SEQ ID NO: 3) andmethods for its recombinant expression are disclosed in U.S. Pat. No.5,439,818 (Fiddes) entitled “DNA Encoding Human Recombinant BasicFibroblast Growth Factor,” which issued on Aug. 08, 1995 (see FIG. 4therein), and in U.S. Pat. No. 5,514,566 (Fiddes), entitled “Methods ofProducing Recombinant Fibroblast Growth Factors,” which issued on May07, 1996 (see FIG. 4 therein). The amino acid sequence of bFGF-2 (SEQ IDNO: 5) and various methods for its recombinant expression are disclosedin U.S. Pat. No. 5,155,214, entitled “Basic Fibroblast Growth Factor,”which issued on Oct. 13, 1992. When the 146 residue forms of hFGF-2 andbFGF-2 are compared, their amino acid sequences are nearly identicalwith only two residues that differ. In particular, in going from hFGF-2to bFGF-2, the sole differences occur at residue positions 112(Thr→Ser)and 128(Ser→Pro).

[0030] FGF-3: FGF-3 (SEQ ID NO: 7) was first identified as an expressionproduct of a mouse int-2 mammary tumor and its amino acid sequence isdisclosed in Dickson et al., “Potential Oncogene Product Related toGrowth Factors,” Nature 326:833 (Apr. 30, 1987). FGF-3 (SEQ ID NO: 7),which has 243 residues when the N-terminal Met is excluded, issubstantially longer than both FGF-1 (human and bovine) and FGF-2(humanand bovine). A comparison of amino acid residues for mFGF-3(SEQ ID NO:7) relative to bFGF-1(SEQ ID NO: 2) and bFGF-2(SEQ ID NO: 5) ispresented in overlap fashion in Dickson et al. (1987). When the aminoacid sequence of mFGF-3(SEQ ID NO: 7) is compared to bFGF-1(SEQ ID NO2): and bFGF-2(SEQ ID NO: 5), FGF-3has 5 locations containing residueinserts relative to both FGF-1and FGF-2. The most significant of theseinserts is a 12 and 14 residue insert relative to FGF-2and FGF-1,respectively, beginning at residue position 135 of FGF-3. Allowing forthe inserts, Dickson discloses that mFGF-3 has 53 residue identitiesrelative to FGF-1 and 69 residue identities relative to FGF-2. Inaddition, FGF-3 contains a hydrophobic N-terminal extension of 10residues relative to the N-terminus of the signal sequence in both FGF-1and FGF-2. Relative to the C-terminus of bFGF-1 and bFGF-2, mFGF-3contains an approximately 60 residue extension. It is unlikely that theC-terminal extension of mFGF-3 is necessary for activity. More likely,it is a moderator of activity by conferring receptor specificity on theFGF.

[0031] FGF-4: The amino acid sequence for the hst protein, now known ashFGF-4 (SEQ ID NO: 8), was first disclosed by Yoshida et al., “GenomicSequence of hst, a Transforming Gene Encoding a Protein Homologous toFibroblast Growth Factors and the int-2-Encoded Protein,” PNAS USA,84:7305-7309 (October 1987). Including its leader sequence, hFGF-4 (SEQID NO: 8) has 206 amino acid residues. When the amino acid sequences ofhFGF-4 (SEQ ID NO: 7), hFGF-1 (SEQ ID NO: 1), hFGF-2 (SEQ ID NO: 3), andmFGF-3 (SEQ ID NO: 7) are compared, residues 72-204 of hFGF- 4 have 43%homology to hFGF- 2 (SEQ ID NO: 5); residues 79-204 have 38% homology tohFGF-I (SEQ ID NO: 1); and residues 72-174 have 40% homology to mFGF- 3(SEQ ID NO: 7). A comparison of these four sequences in overlap form isshown in Yoshida (1987) at FIG. 3. Further, the Cys at residue positions88 and 155 of hFGF- 4 are highly conserved among hFGF-1, hFGF- 2, mFGF-3, and hFGF- 4 and are found in a homologous region.

[0032] The two putative cell binding sites of hFGF- 2 (SEQ ID NO: 3)occur at residue positions 36-39 and 77-81 thereof. See Yoshida (1987)at FIG. 3. The two putative heparin binding sites of hFGF- 2 occur atresidue positions 18-22 and 107-111 thereof. See Yoshida (1987) at FIG.3. Given the substantial similarity between the amino acid sequences forhuman and bovine FGF- 2, we would expect the cell binding sites forbFGF- 2 (SEQ ID NO: 5) to also be at residue positions 36-39 and 77-81thereof, and the heparin binding sites to be at residue positions 18-22and 107-111 thereof. In relation to hFGF- I (SEQ ID NO: 1), the putativecell binding sites occur at residues 27-30 and 69-72, and the putativeheparin binding sites occur at residues 9-13 and 98-102. Insofar asbFGF-1(SEQ ID NO: 2) has the identical amino acids at residue positions9-13, 27-30, 69-72, and 98-102 as does hFGF-l (SEQ ID NO: 1), bFGF-1would be expected to have the same cell and heparin binding sites asdoes hFGF- 1.

[0033] FGF- 5: The amino acid sequence and method for cloning hFGF- 5(SEQ ID NO: 15) are disclosed in Zhan, et al., “The Human FGF-5 OncogeneEncodes a Novel Protein Related to Fibroblast Growth Factors,” Molec.and Cell. Biol., 8( 8): 3487-3495 (August 1988). The Applicants alsosequenced the FGF- 5 and obtained the amino acid sequence of SEQ ID NO:9, which differed from Zhan's sequence at residue position 236 (having aLys instead of the Zhan's Asn) and at residue position 243 (having a Proinstead of Zhan's Ser). Both hFGF- 5 (SEQ ID NO: 9) and hFGF- 5 (SEQ IDNO: 15) have 266 amino acid residues that include a leader sequence of67 residues upstream of the first residue of the FGF- 2 of SEQ ID NO: 5and a tail sequence that extends about 47 residues beyond the C-terminusof hFGF- 2. A comparison between the amino acid sequences of hFGF-1(SEQID NO: 1), hFGF- 2 (SEQ ID NO: 3), mFGF- 3 (SEQ ID NO: 7), hFGF- 4 (SEQID NO: 8), and FGF- 5 (SEQ ID NO: 9) is presented in FIG. 2 of Zhan(1988). In FIG. 2 of Zhan, hFGF- 1, hFGF- 2, mFGF- 3, and hFGF- 4 areidentified as aFGF (i.e., acidic FGF), bFGF (i.e., basic FGF), int- 2,and hstKS 3, respectively, i.e., by their original names. In the abovereferenced comparison, two blocks of FGF- 5 amino acid residues (90 to180 and 187-207) showed substantial homology to FGF 1-4, i.e., 50.4%with FGF-4, 47.5% with FGF-3, 43.4% with FGF-2 and 40.2% with hFGF-1.See Zhan (1988) at FIG. 2. U.S. Pat. Nos. 5,155,217 (Goldfarb) and5,238,916 (Goldfarb), which correspond to the Zhan publication, refer tothe FGF-5 of Zhan as FGF-3. However, the art (as evidenced by Coulierbelow) has come to recognize that the hFGF of Zhan (and Goldfarb) asFGF-5 and not as FGF-3. The two Goldfarb patents contain the same aminoacid sequence for hFGF-5 (SEQ ID NO: 15) as reported above by Zhan.

[0034] FGF-6: The amino acid sequence and method for cloning hFGF-6 (SEQID NO: 10) are disclosed in Coulier et al., “Putative Structure of theFGF-6 Gene Product and Role of the Signal Peptide,” Oncogene 6:1437-1444(1991). hFGF-6 is one of the largest of the FGFs, having 208 amino acidresidues. When the amino acid sequences of human FGF-1, FGF-2, FGF-3,FGF-4, FGF-5, FGF-6, and FGF-7 are compared, there are strongsimilarities in the C-terminal two-thirds of the molecules(corresponding e.g., to residues 78-208 of hFGF-6 (SEQ ID NO: 10)). Inparticular, 23 residues, including two cysteines (at positions 90-157 ofhFGF-6 of SEQ ID NO: 10) were identical between the seven members of thefamily. This number increases to 33 residues when conserved amino acidresidues are considered. The overall similarities between these sevenhuman FGFs range from 32 to 70% identical residues and 48 to 79%conserved residues for the C-terminal two-thirds of the molecules. Thesequence comparisons, relative to FGF-6, are shown in Table 1 herein.TABLE 1 Amino Acid Sequence Comparison of hFGF-6 With Other hFGFsIdentical Conserved Identical Conserved SEQ ID NO: Residues* Residues**Residues* (%) Residues** (%) hFGF-4 8 91 103 70 79 hFGF-5 9 64 82 49 63hFGF-3 7 55 78 42 60 hFGF-2 3 54 69 42 53 hFGF-7 11 47 68 36 52 hFGF-1 142 62 32 48

[0035] Referring to Table 1, FGF-6 has the highest correspondence (91identical residues/103 conserved residues) with FGF-4. This amounts to70% identical and 79% conserved residues. hFGF-6 (SEQ ID NO: 10)differed most from hFGF-3 (SEQ ID NO: 2); hFGF-2 (SEQ ID NO: 3), hFGF-7(SEQ ID NO: 11), and FGF-1 (SEQ ID NO: 1), with 42, 42, 36, and 32identical residues, respectively.

[0036] An overlayed comparison of the amino acid sequences of FGFs 1-7is shown in FIG. 3 of incorporated Coulier (1991). FIG. 3 of Couliershows that when the C-terminal two thirds of the FGF molecules arealigned, there are 23 residue positions wherein the residues from allseven FGF members are identical. There are also ten residue positionswherein residues from all seven FGF members are conserved. Coulier(1991) at FIG. 3. In combination, these identical and conserved residuesform about 6 locations of three to five residues on the terminal twothirds of each of the FGFs 1-7, wherein three to five residues aregrouped together in all seven species of human FGF (i.e., hFGF 1-7).

[0037] FGF-7: The amino acid sequence of hFGF-7 (SEQ ID NO: 11) isdisclosed in Miyamoto, et al., “Molecular Cloning of a Novel CytokinecDNA Encoding the Ninth Member of the Fibroblast Growth Factor Family,Which Has a Unique Secretion Property,” Mol. and Cell. Biol.13(7):4251-4259 (1993). In Miyamoto, the hFGF-7 was referred to by itsolder name KGF. As reflected in SEQ ID NO: 11, FGF-7 has 191 amino acidresidues. Miyamoto compared hFGF-7 (SEQ ID NO: 11) to hFGF 1-6 andhFGF-9 shows that the carboxy terminal two thirds of the FGF-7 hascomparable homology with the distal two thirds of the other members ofthe group. See Miyamoto (1993) at page 4254 (FIG. 2).

[0038] FGF-8: The amino acid sequence of mFGF-8 (SEQ ID NO: 12) and amethod for its recombinant expression are disclosed in Tanaka et al.,“Cloning and Characterization of an Androgen-induced Growth FactorEssentialfor the Growth of Mouse Mammary Carcinoma Cells,” PNAS USA,89:8928-8932 (1992). The mFGF-8 of Tanaka has 215 amino acid residues.MacArthur, et al., “FGF-8 Isoforms Activate Receptor Splice Forms thatAre Expressed in Mesenchymal Regions of Mouse Development,” Development,121:3603-3613 (1995) discloses that FGF-8 has 8 different isoforms thatdiffer at the mature N-terminus but that are identical over theC-terminal region. The 8 isoforms arise because FGF-8 has 6 exons ofwhich the first four (which correspond to the first exon of most otherFGF genes) result in alternative splicing.

[0039] FGF-9: The amino acid sequence of hFGF-9 and a method for itsrecombinant expression are disclosed in Santos-Ocampo, et al.,“Expression and Biological Activity of Mouse Fibroblast Growth Factor,”J. Biol. Chem., 271(3):1726-1731 (1996). Notwithstanding its title,Ocampo discloses the amino acid sequence of both hFGF-9 (SEQ ID NO: 13)and mFGF-9. Both the human and murine FGF-9 molecules have 208 aminoacid residues and sequences that differ by only two residues. Inparticular, the hFGF-9 has Asn and Ser at residues 9 and 34,respectively, whereas the mFGF-9 has Ser and Asn, respectively. FGF-9has complete preservation of the conserved amino acids that define theFGF family. Santos-Ocampo (1996) at page 1726. Half-maximal activationof FGF-9 is seen at 185 ng/ml heparin, whereas half-maximal activationof FGF-1 is seen at 670 ng/ml heparin. Santos-Ocampo (1996) at page1730. When compared to FGF-1, both FGF-2 and FGF-9 require lower heparinconcentrations for optimal activity.

[0040] FGF-98: The amino acid sequence of hFGF-98 (SEQ ID NO: 14) and amethod for its recombinant expression are disclosed in provisionalpatent application Serial No. 60/083,553 which is hereby incorporatedherein by reference in its entirety. hFGF-98, which is also known ashFGF-18, has 207 amino acid residues. Thus, hFGF-6 (207 residues),hFGF-9 (208 residues), and hFGF-98 (207 residues) are similar in size.FGFs differentially bind to and activate one or more of four relatedtransmembrane receptors which in turn mediate a biological response. TheFGF receptors (“FGFR”) are members of the tyrosine kinase receptorsuperfamily. The extracellular domain of the FGFR comprises 2-3immunoglobulin-like (“IG-like”) domains that are differentiallyexpressed as a result of alternative splicing. Another alternativesplicing event can also alter the sequence of the carboxy-terminal halfof the Ig-like domain Imf without altering the reading frame.Santos-Ocampo (1996). The two splice forms, which are referred to as “b”and “c”, occur for FGFRs 1,2,3 but not 4. A more detailed description ofthe FGFR is found in Mathieu, et al, “Receptor Binding and MitogenicProperties of Mouse Fibroblast Growth Factor 3,” J. Biol. Chem.,270(41):24197-24203 (1995). The ability of FGF 1-9 to differentiallystimulate FGFRs was receptor dependent as reported by Ornitz et al., J.Biol. Chem., 271(25):15292-15297 (1996). In Ornitz, the cell line BaF3was divided into fractions and each fraction was transfected to expressone of the following FGF receptors: FGFR1b, FGFR1c, FGFR2b, FGFR2c,FGFR3b, FGFR3c, and FGF4 (minus one Ig-like domain). Thereafter, thetransformed cell lines were exposed to one of FGF1-9 (5 nM) and heparin(2 μg/ml) as a cofactor. The mitogenic response was then measured byincorporation of [³H] thymidine. The results in cpm are as follows:

[0041] 1. FGFR1b: similar mitogenic responses were produced by hFGF-1(32,000 cpm) and hFGF-2 (28,000 cpm) with the next highest responses bymFGF-3 (about 16,000 cpm) and hFGF-4 (15,000 cpm);

[0042] 2. FGFR1c: similar mitogenic responses were produced by hFGF-1,hFGF-2, hFGF-4, hFGF-5, and hFGF-6 (about 36,000 cpm), with mFGF-9producing the only other significant response (about 19,000 cpm);

[0043] 3. FGFR2b: best mitogenic responses were by hFGF-7 (14,000 cpm),hFGF-1(12,500 cpm), and mFGF-3 (9,500 cpm);

[0044] 4. FGFR 2c: best mitogenic responses were by hFGF-4 (21,000 cpm),mFGF-9 (20,000 cpm), hFGF-6 (16,500 cpm), hFGF-1 (16,000 cpm), hFGF-2(14,500 cpm), hFGF-5 (9,500 cpm), and mFGF-8 (9,000 cpm);

[0045] 5. FGFR 3b: mitogenic responses only by hFGF-1 (37,000 cpm) andmFGF-9 (26,000 cpm);

[0046] 6. FGFR3c: best mitogenic responses by hFGF-1 (39,000 cpm),hFGF-2 (34,000 cpm), hFGF-4 (33,000 cpm), mFGF-8 (32,500 cpm), mFGF-9(31,000 cpm), hFGF-5 (16,000 cpm), and hFGF-6 (13,000 cpm);

[0047] 7. FGFR4A: best mitogenic responses by hFGF-2 (29,000 cpm),hFGF-4 and hFGF-6 (27,000 cpm), mFGF-8 (25,000 cpm), mFGF-1 (24,000cpm), and hFGF-9 (20,000 cpm) with all others being 6,000 cpm or less.

[0048] As reflected above, only FGF-1 induces a significant mitogenicresponse in all of the receptors tested. Thus, FGF-1 can be thought ofas a universal ligand with N-and C-terminal additions to the moleculegiving rise to receptor specificity associated with the other FGF. Giventhe potential for diverse responses in vivo by systemically administeredFGF, the present invention minimizes the potential for systemicresponses by localized administration, and by discovering theappropriate dosage for the localized administration, i.e., byadministering a therapeutically effective amount of an FGF into at leastone coronary artery of a patient in need of treatment for CAD.

[0049] In the Examples that follow, bFGF-2 (SEQ ID NO: 5) wasadministered in vivo to rats, pigs, and humans, and tested forangiogenic activity. The bFGF-2 of the Examples was made as described inU.S. Pat. No. 5,155,214. In the '214 patent, a DNA of SEQ ID NO: 4,which encodes a bFGF (hereinafter “FGF-2”) of SEQ ID NO: 5, is insertedinto a cloning vector, such as pBR322, pMB9, Col E1, pCRI, RP4 orX-phage, and the cloning vector is used to transform either a eukaryoticor prokaryotic cell, wherein the transformed cell expresses the FGF-2.In one embodiment, the host cell is a yeast cell, such as Saccharomycescerevisiae. The resulting full-length FGF-2 that is expressed has 146amino acids in accordance with SEQ ID NO: 5. Although the FGF-2 of SEQID NO: 5 has four cysteines, i.e., at residue positions 25, 69, 87,and92, there are no internal disulfide linkages. ['214 at col. 6, lines59-61.] However, in the event that cross-linking occurred underoxidative conditions, it would likely occur between the residues atpositions 25 and 69.

[0050] The FGF-2 of SEQ ID NO: 5, which is of bovine origin, like thecorresponding human FGF-2 is initially synthesized in vivo as apolypeptide having 155 amino acid residues. Abraham et al. “Human BasicFibroblast Growth Factor: Nucleotide Sequence and Genomic Organization,”EMBO J., 5(10):2523-2528 (1986). When compared to the full-length155-residue bovine FGF-2 of Abraham, Applicants' FGF-2 of SEQ ID NO: 5lacks the first nine amino acid residues, Met Ala Ala Gly Ser Ile ThrThr Leu (SEQ ID NO: 3), at the N-terminus of the correspondingfull-length molecule. As discussed above, the FGF-2 of SEQ ID NO: 5differs from human FGF-2 in two residue positions. In particular, theamino acids at residue positions 112 and 128 of the bFGF-2 of SEQ ID NO:5 are Ser and Pro, respectively, whereas in hFGF-2, they are Thr andSer, respectively. Given this substantial structural identity, the invivo clinical results provided in the Examples and discussed elsewhereherein on bFGF-2 (SEQ ID NO: 5) should be directly applicable to hFGF-2(SEQ ID NO: 3).

[0051] The recombinant bFGF-2 (SEQ ID NO: 5) of the Examples waspurified to pharmaceutical quality (98% or greater purity) using thetechniques described in detail in U.S. Pat. No. 4,956,455, entitled“Bovine Fibroblast Growth Factor” which issued on Sep. 11, 1990 andwhich was incorporated herein by reference in its entirety. Inparticular, the first two steps employed in the purification of therecombinant bFGF-2 of Applicants' unit dose are “conventionalion-exchange and reverse phase HPLC purification steps as describedpreviously.” [U.S. Pat. No. 4,956,455, citing to Bolen et al., PNAS USA81:5364-5368 (1984).] The third step, which the '455 patent refers to asthe “key purification step”['455 at col. 7, lines 5-6], isheparin-SEPHAROSER® affinity chromatography, wherein the strong heparinbinding affinity of the FGF-2 is utilized to achieve severalthousand-fold purification when eluting at approximately 1.4 M and 1.95M NaCl ['455 at col. 9, lines 20-25]. Polypeptide homogeneity wasconfirmed by reverse-phase high pressure liquid chromatography(RP-HPLC). Buffer exchange was achieved by SEPHADEX® G-25(M) gelfiltration chromatography.

[0052] In addition to the FGF of any one of SEQ ID NOS: 1-3, 5, 8-10, or12-14, the active agent in the unit dose of the present invention alsocomprises an “angiogenically active fragment thereof.” By the term“angiogenically active fragment thereof” is meant a fragment of any oneof the FGF of SEQ ID NOS: 1-3, 5, 8-10, or 12-14 that has about 80% ofthe residues sequence of SEQ ID NO: 5 and that retains the angiogeniceffect of the corresponding mature FGF. A common truncation is theremoval of the N-terminal methionine, using well known techniques suchas treatment with a methionine aminopeptidase. A second desirabletruncation comprises the FGF without its leader sequence. Those skilledin the art recognize the leader sequence as the series of hydrophobicresidues at the N-terminus of a protein that facilitate its passagethrough a cell membrane but that are not necessary for activity and thatare not found on the mature protein.

[0053] Preferred truncations are determined relative to hFGF-2 (or theanalogous bFGF-2). As a general rule, the amino acid sequence of an FGFis aligned with FGF-2 to obtain maximum homology. Portions of the FGFthat extend beyond the corresponding N-terminus of the aligned FGF-2(SEQ ID NO: 3) are suitable for deletion without adverse effect.Likewise, portions of the FGF that extend beyond the C-terminus of thealigned FGF-2 (SEQ ID NO: 3) are also capable of being deleted withoutadverse effect.

[0054] Fragments of FGF that are smaller than those described above arealso within the scope of the present invention so long as they retainthe cell binding portions of FGF and at least one heparin bindingsegment. As already discussed above, the heparin binding segments ofFGF-2 (human or bovine) occur at residues 18-22 and 107-111, whereas thecell binding portions occur at residues 36-39 and 77-81. For example, itis well known in the art that N-terminal truncations of bFGF-2 do noteliminate its angiogenic activity in cows. In particular, the artdiscloses several naturally occurring and biologically active fragmentsof bFGF-2 of SEQ ID NO: 5 that have N-terminal truncations relative tothe bFGF-2 of SEQ ID NO: 5. An active and truncated bFGF-2 havingresidues 12-146 of SEQ ID NO: 5 was found in bovine liver and anotheractive and truncated bFGF-2, having residues 16-146 of SEQ ID NO: 5 wasfound in the bovine kidney, adrenal glands, and testes. [See U.S. Pat.No. 5,155,214 at col. 6, lines 41-46, citing to Ueno, et al., Biochemand Biophys Res. Comm., 138:580-588 (1986).] Likewise, other fragmentsof the bFGF-2 of SEQ ID NO: 5 that are known to have FGF activity areFGF-2 (24-120)-OH and FGF-2 (30-110)-NH₂. [U.S. Pat. No. 5,155,214 atcol. 6, lines 48-52.] These latter fragments retain both of the cellbinding portions of bFGF-2 (SEQ ID NO: 5) and one of the heparin bindingsegments (residues 107-111). Accordingly, the angiogenically activefragments of an FGF typically encompass those terminally truncatedfragments of an FGF that when aligned to an FGF-2 to maximize homology,have at least residues that correspond to residues 30-110 of bFGF-2 ofSEQ ID NO: 5 (or the hFGF-2 of SEQ ID NO: 3); more typically, at leastresidues that correspond to residues 18-146 of bFGF-2 of SEQ ID NO: 5.

[0055] The unit dose of the present invention also comprises an“angiogenically active . . . mutein” of the FGF of any one of SEQ IDNOS: 1-3, 5, 8-10, or 12-14. By the term “angiogenically active . . .mutein” is meant a mutated form of the FGF of any one of SEQ ID NOS:1-3, 5, 8-10, or 12-14 that structurally retains at least 80%,preferably 90%, of the residues of any one of SEQ ID NOS: 1-3, 5, 8-10,or 12-14 in their respective positions, and that functionally retainsthe angiogenic activity of the FGF of any one of SEQ ID NOS: 1-3, 5,8-10, or 12-14. Preferably, the mutations are “conservativesubstitutions” using L-amino acids, wherein one amino acid is replacedby another biologically similar amino acid. Examples of conservativesubstitutions include the substitution of one hydrophobic residue suchas Ile, Val, Leu, Pro, or Gly for another, or Phe⇄Tyr, Ser⇄Thr, or thesubstitution of one polar residue for another, such as between Arg andLys, between Glu and Asp, or between Gln and Asn, and the like.Generally, the charged amino acids are considered interchangeable withone another. However, to make the substitution more conservative, onetakes into account both the size and the likeness of the charge, if any,on the side chain. Other suitable substitutions include the substitutionof serine for one or both of the cysteines at residue positions whichare not involved in disulfide formation, such as residues 87 and 92 inhFGF-2 (SEQ ID NO: 3) or bFGF-2 (SEQ ID NO: 5). Preferably,substitutions are introduced at the N-terminus, which is not associatedwith angiogenic activity. However, as discussed above, conservativesubstitutions are suitable for introduction throughout the molecule.

[0056] One skilled in the art, using art known techniques, is able tomake one or more point mutations in the DNA encoding an FGF of any oneof SEQ ID NOS: 1-3, 5, 8-10, or 12-14 to obtain expression of an FGFpolypeptide mutein (or fragment mutein) having angiogenic activity foruse within the unit dose, compositions and method of the presentinvention. To prepare an angiogenically active mutein of the FGF of anyone of SEQ ID NOS: 1-3, 5, 8-10, or 12-14, one uses standard techniquesfor site-directed mutagenesis, as known in the art and/or as taught inGilman, et al., Gene, 8:81 (1979) or Roberts, et al., Nature, 328:731(1987), to introduce one or more point mutations into the CDNA thatencodes the FGF of any one of SEQ ID NOS: 1-3, 5, 8-10, or 12-14.

[0057] In a second aspect, the present invention is directed to apharmaceutical composition comprising a safe and an angiogenicallyeffective dose of an FGF of any one of SEQ ID NOS: 1-3, 5, 8-10, or12-14 or an angiogenically active fragment or mutein thereof, and apharmaceutically acceptable carrier. Typically, the safe andangiogenically effective dose of the pharmaceutical composition of thepresent invention is in a form and a size suitable for administration toa human patient and comprises (i) 0.2 μg/kg to 36 μg/kg of an FGF of anyone of SEQ ID NOS: 1-3, 5, 8-10, or 12-14 or an angiogenically activefragment or mutein thereof, (ii) and a pharmaceutically acceptablecarrier. In other embodiments, the safe and angiogenically effectivedose comprises 0.2 μg/kg to 2 μg/kg, 2 μg/kg to 20 μg/kg, or 20 μg/kg to36 μg/kg of the FGF of any one of SEQ ID NOS: 1-3, 5, 8-10, or 12-14 oran angiogenically active fragment or mutein thereof, and apharmaceutically acceptable carrier.

[0058] By the term “pharmaceutically acceptable carrier” as used hereinis meant any of the carriers or diluents known in the art for thestabilization and/or administration of a proteinaceous medicament, suchas the FGF of any one of SEQ ID NOS: 1-3, 5, 8-10, or 12-14 disclosedherein, that does not itself induce the production of antibodies harmfulto the individual receiving the composition, and which may beadministered without undue toxicity. Within another aspect of theinvention, pharmaceutical compositions are provided, comprising arecombinant FGF of any one of SEQ ID NOS: 1-3, 5, 8-10, or 12-14 or anangiogenically active fragment or mutein thereof in combination with apharmaceutically acceptable carrier or diluent. Such pharmaceuticalcompositions may be prepared either as a liquid solution, or as a solidform (e.g., lyophilized) which is dissolved in a solution prior toadministration. In addition, the composition may be prepared withsuitable carriers or diluents for IC injection or administration.Pharmaceutically acceptable carriers or diluents are nontoxic to a humanrecipient at the dosages and concentrations employed. Representativeexamples of suitable carriers or diluents for injectable or infusiblesolutions include sterile water or isotonic saline solutions, which arepreferably buffered at a suitable pH (such as phosphate-buffered salineor Tris-buffered saline), and optionally contain mannitol, dextrose,glycerol, ethanol, and/or one or more polypeptides or proteins such ashuman serum albumin (HSA). Stabilizers, such as trehalose, thioglycerol,and dithiothreitol (DTT), may also be added.

[0059] A typical pharmaceutical composition comprises 0.001 to 10μmg/ml, more typically 0.03 to 0.5 μmg/ml, of a rFGF of any one of SEQID NOS: 1-3, 5, 8-10, or 12-14 or an angiogenically active fragment ormutein thereof, 10 mM thioglycerol, 135 mM NaCl, 10 mM Na citrate, and 1mM EDTA, pH 5. A suitable diluent or flushing agent for the abovedescribed composition is any of the above describeD carriers. Typically,the diluent is the carrier solution itself comprising 10 mMthioglycerol, 135 mM NaCI, 10 mM Na citrate, and 1 mM EDTA, pH 15. TherFGF of any one of SEQ ID NOS: 1-3, 5, 8-10, or 12-14 or anangiogenically active fragment or mutein thereof is unstable for longperiods of time in liquid form. To maximize stability and shelf life,the pharmaceutical composition of the present invention comprising aneffective amount of rFGF of any one of SEQ ID NOS: 1-3, 5, 8-10, or12-14 or an angiogenically fragment or mutein thereof, in apharmaceutically acceptable aqueous carrier should be stored frozen at−60° C. When thawed, the solution is stable for 6 months at refrigeratedconditions. A typical unit dose would comprise about 5-10 ml of theabove described composition having 1.5-8 mg of the FGF of any one of SEQID NOS: 1-3, 5, 8-10, or 12-14.

[0060] In another embodiment, the pharmaceutical composition comprises aunit dose of FGF of any one of SEQ ID NOS: 1-3, 5, 8-10, or 12-14 or anangiogenically active fragment or mutein thereof in lyophilized(freeze-dried) form. In this form, the unit dose of FGF would be capableof being stored at refrigerated temperatures for substantially longerthan 6 months without loss of therapeutic effectiveness. Lyophilizationis accomplished by the rapid freeze drying under reduced pressure of aplurality of vials, each containing a unit dose of the FGF of thepresent invention therein. Lyophilizers, which perform the abovedescribed lyophilization, are commercially available and readilyoperable by those skilled in the art. Prior to administration to apatient, the lyophilized product is reconstituted to a knownconcentration, preferably in its own vial, with an appropriate sterileaqueous diluent, typically 0.9% (or less) sterile saline solution, or acompatible sterile buffer, or even sterile deionized water. Dependingupon the weight of the patient in kg, a single dose comprising from 0.2μg/kg to 36 μg/kg of the FGF of any one of SEQ ID NOS: 1-3, 5, 8-10, or12-14 or an angiogenically active fragment or mutein thereof iswithdrawn from the vial as reconstituted product for administration tothe patient. Thus, an average 70 kg man that is being dosed at 24 μg/kg,would have a sufficient volume of the reconstituted product withdrawnfrom the vial to receive an IC infusion of (70 kg ×24 μg/kg) 1680 μg(i.e., 1.680 mg).

[0061] In its third aspect, the present invention is directed to amethod for treating a patient in need of treatment for CAD or MI, usingthe above described unit dose or pharmaceutical composition to treat ahuman patient for coronary artery disease (CAD). In particular, thepresent invention is directed to a method for treating a human patientfor coronary artery disease, comprising administering a safe andtherapeutically effective amount of a recombinant FGF of any one of SEQID NOS: 1-3, 5, 8-10, or 12-14 or an angiogenically active fragment ormutein thereof to one or more, typically two, coronary vessels of ahuman patient in need of treatment for coronary artery disease. Apreferred coronary vessel is the coronary artery, although graftedsaphenous veins and grafted internal mammary arteries, as provided bycoronary angioplasty, are also suitable.

[0062] The method of the present invention provides clinical treatmentof the underlying condition (i.e., CAD or MI) and not merely a treatmentof the symptoms, such as provided by nitrates. Typically, the safe andtherapeutically effective amount of the method of the present inventioncomprises 0.2 μg/kg to 36 μg/kg of the FGF of any one of SEQ ID NOS:1-3, 5, 8-10, or 12-14 or an angiogenically active fragment or muteinthereof in a pharmaceutically acceptable carrier. In other embodiments,the safe and therapeutically effective amount comprises 0.2 μg/kg to 2μg/kg, 2 μg/kg to 20 μg/kg, or 20 μg/kg to 36 μ/kg of the FGF of any oneof SEQ ID NOS: 1-3, 5, 8-10, or 12-14 or an angiogenically fragment ormutein thereof in a pharmaceutically acceptable carrier. In absoluteterms, the safe and therapeutically effective amount is about 0.008 mgto about 6.1 mg of the FGF of any one of SEQ ID NOS: 1-3, 5, 8-10, or12-14 or an angiogenically fragment or mutein thereof, more typically,0.3 mg to 3.5 mg of the FGF of any one of SEQ ID NOS: 1-3, 5, 8-10, or12-14 or an angiogenically fragment or mutein thereof.

[0063] The therapeutically effective amount of the rFGF-2 of the presentinvention is administered to at least one coronary vessel of a humanpatient diagnosed with CAD, symptomatic despite optimal medicalmanagement, using standard cardiac catheterization techniques alreadyknown and used in the art for the intracoronary administration ofmedicaments, e.g., thrombolytics, streptokinase, or radio-opaque dyes ormagnetic particles used to visualize the coronary arteries. By way ofexample, a coronary catheter is inserted into an artery (e.g., femoralor subclavian) of the patient in need of treatment and the catheter ispushed forward, with visualization, until it is positioned in theappropriate coronary vessel of the patient to be treated. Using standardprecautions for maintaining a clear line, the pharmaceutical compositionin solution form is administered by infusing the unit dose substantiallycontinuously over a period of 10 to 30 minutes. Although thepharmaceutical composition of the invention could be administered over alonger period of time, the Applicants perceive no benefit and apotentially increased risk of thrombosis in doing so. Typically, aportion (e.g., one half) of the unit dose is administered in a firstcoronary vessel. Then, the catheter is repositioned into a secondsecondary coronary vessel and the remainder of the unit dose isadministered with flushing of the catheter. Using the above-describedrepositioning procedure, portions of the unit dose may be administeredto a plurality of coronary vessels until the entire unit dose has beenadministered. After administration, the catheter is withdrawn usingconventional art known protocols. Signs of coronary angiogenesis areapparent in a matter of days following IC administration of the unitdose. Therapeutic benefit is seen as early as two weeks following the ICFGF administration. Clinically significant improvement is readilydemonstrable by objective criterion (ETT and/or SAQ) 30 days followingIC administration of the unit dose. In certain patients with progressiveCAD disease, it may be necessary or appropriate to administer a unitdose of the FGF, for example, every six months or annually, to overcomethe progression of the CAD during that interim period.

[0064] One of the benefits of the method of the present invention iscardiac angiogenesis. Accordingly, in another aspect, the presentinvention is directed to a method for inducing angiogenesis in a heartof a human patient, comprising administering as a unit dose into one ormore coronary vessels of a human patient in need of coronaryangiogenesis about 0.2 μg/kg to about 36 μg/kg (or in absolute termsabout 0.008 mg to about 6.1 mg) of a recombinant FGF of any one of SEQID NOS: 1-3, 5, 8-10, or 12-14 or an angiogenically active fragment ormutein thereof.

[0065] Fifty-two (52) human patients diagnosed with CAD, who satisfiedthe criteria of Example 2 herein, were administered a unit dose of 0.33μg/kg to 48 μg/kg of the FGF-2 of SEQ ID NO: 5 by IC infusion over abouta 20 minute period. The 52 treated patients were then assessed by theSeattle Angina Questionnaire, which provides an assessment based upon amixed combination of objective and subjective criteria. See Table 2. TheSeattle Angina Questionnaire is a validated, disease-specific instrumentwith the following five subscales that are assessed both before andafter treatment: 1) “exertional capacity”=limitation of physicalactivity; 2) “disease perception”=worry about MI; 3) “treatmentsatisfaction”; 4) “angina frequency”=number of episodes and sublingualnitroglycerin usage; and 5) “angina stability”=number of episodes withmost strenuous physical activity. The possible range for each of thefive subscales is 0 to 100 with the higher scores indicating a betterquality of life. Moreover, a mean change of 8 points or more between themean baseline scores (before treatment) and the post-treatment scores isrecognized as being “clinically significant.” Table 2 reports that the28 patients, who were pretested and then administered a single unit doseof 0.33 μg/kg to 24 μg/kg of the FGF-2 of SEQ ID NO: 5 by IC infusion,exhibited a mean score increase of 13 to 36 points for the five “qualityof life” criteria assessed by the “Seattle Angina Questionnaire.” SeeTable 2 herein. These 13 to 36 point increases were about 1.6 to 4.5times greater than the 8 point change which is recognized in the art asbeing “clinically significant” in alternative modes of treatment. SeeTable 2 herein. Moreover, when the combined results for the first 15patients of Table 2 were broken down between low dose (less than orequal to 2 μg/kg) and high (more than 2 μg/kg) doses of FGF-2 of SEQ IDNO: 5, and assessed by the “Seattle Angina Questionnaire,” both doseswere found to provide increased scores that ranged from about 12.3 to58.1 and about 10.9 to 32.1, respectively. See Table 3 herein. Theincreased scores were about 1.4 to 7.2 times greater than the 8 pointchange which is considered to be “clinically significant” in alternativemodes of treatment.

[0066] In the same Phase I trial, fifty two human patients who werediagnosed with CAD and who satisfied the criteria of Example 2 herein,were administered IC a TABLE 2 COMPARISON OF QUALITY OF LIFE BEFORE AND57 DAYS AFTER IC FGF-2 Baseline Seattle Angina (Pre FGF-2) 57 Days PostQuestionnaire Mean FGF-2 Mean Mean (SAQ) Subscales Score ± SD Score ± SDChange¹ p Value n Exertional 55 ± 23 68 ± 25 13* 0.02 28 Capacity Angina42 ± 32 66 ± 28 24* <0.001 28 Frequency Angina Stability 46 ± 26 82 ± 2036* <0.001 27 Disease 40 ± 21 61 ± 26 19* <0.001 28 Perception Treatment74 ± 24 88 ± 16 14* 0.002 28 Satisfaction

[0067] TABLE 3 IMPROVEMENTS IN THE QUALITY OF LIFE AT DAY 57 (POST ICrFGF-2) AT LOWER AND HIGHER DOSES Dose <2 μg/kg Dose <2 μg/kg IC rFGF-2IC rFGF-2 (n = 7) (n = 8) Mean Change Mean Change Seattle Angina inScore in Score Questionnaire (Day 57 score- (Day 57 score- Independent(SAQ) Subscales screen score) screen score) Samples t-test Exertional12.30 (23.3) 15.98 (28.7) t = −.27 p = .79 Capacity Disease 26.19 (26.9)24.47 (21.2) t = .14 p = .89 Perception Treatment 22.32 (27.7) 10.93(17.3) t = .97 p = .35 Satisfaction Angina 28.57 (27.3) 13.75 (22.6) t =1.15 p = .27 Frequency Angina Stability 58.13 (12.9) 32.14 (34.5) t =1.75 p = .108

[0068] single unit dose of 0.33 μg/kg to 48 μg/kg of FGF-2 of SEQ ID NO:5. The maximum tolerated dose (MTD) in humans was defined as 36 μg/kgbased upon the occurrence of severe but transient hypotension in 2/10patients at 48 μg/kg. (In contrast, the MTD in pigs was defined as 6.5μg/ml.) At one of the sites, the hearts of 23 human patients wereassessed both before (“baseline”) and 30 and 60 days after treatment bymagnetic resonance imaging (MRI) for objective signs of improvedcoronary sufficiency. Among the objective criteria assessed by MRI arethe following: 1) left ventricular (LV) ejection fraction (EF); 2)normal wall thickness (NWT); 3) normal wall motion (NWM); 4) collateralextent; 5) ischemic area zone; 6) targeted wall thickness (TWT); 7)targeted wall motion (TWM); and 8) perfusion or delayed arrival zone (%LV). The patients were also assessed for angina, treadmill exerciseduration, rest/exercise nuclear perfusion. The results are summarized inTable 4. Table 4 reflects that the baseline angina class decreased from2.6 to 1.4 and 1.2 at 30 and 60 days, respectively post IC FGF-2. Themean treadmill exercise time increased from a baseline of 8.5 minutes to9.4 and 10.0 minutes at 30 and 60 days, respectively, post treatment. Nosignificant difference was observed in the left ventricular ejectionfraction (LV EF). However, the target wall motion increasedsignificantly, moving from a baseline of 15.4% to 23.5% (day 30) and24.1% (day 60) post FGF-2 treatment. Likewise the target wall thickeningincreased significantly from a baseline of 28.7% to 34.7% (day 30) and45.9% (day 60) post FGF-2 treatment. There was also a significantincrease in perfusion, as measured by a decrease in the delayed arrivalzone (% LV), with the delayed arrival zone decreasing from a baseline of18.9% to 7.1% (day 30) and 1.82% (day 60) post FGF-2 treatment. Thus,providing CAD patients with a single IC infusion of FGF-2 in accordancewith the present invention provided the patients with a significantphysical improvement as objectively measured by MRI and otherconventional criteria.

Pharmacokinetics and Metabolism

[0069] The molecular structure of FGF-2 contains a positively chargedtail that is known to bind to proteoglycan chains (heparin andheparin-like structures) on cell surfaces and on the endothelial wall ofthe vasculature. See Moscatelli, et al., “Interaction of BasicFibroblast Growth Factor with Extracellular Matrix and TABLE 4 MEAN DATAAND DATA RESULTS AS A FUNCTION OF TIME AND DOSE Baseline 30 Day 60 DayAngina Class  2.6 ± 0.7  1.4 ± 0.9***  1.2 ± 0.8*** Exercise Time (min.) 8.5 ± 2.6  9.4 ± 1.9*** 10.0 ± 2.5** LV EF (%) 47.4 ± 12.3 47.4 ± 10.648.6 ± 11.0 Target Wall Motion (%) 15.4 ± 10.1 23.5 ± 12.0* 24.1 ±10.1** Target Wall Thickening 28.7 ± 14.0 34.7 ± 14.1 45.9 ± 11.7** (%)Delayed Arrival Zone 18.9 ± 8.3  7.1 ± 3.6*** 1.82 ± 2.4*** (% LV)

[0070]Receptors, ” Ann. NY Acad. Sci., 638:177-181 (1981). Because theendothelium is responsible for binding FGF- 2 and acts as a sink afterinjection, we believed that rFGF-2 will undergo a fast biodistributionphase right after administration. Accordingly, we targeted theintracoronary as opposed to the intravenous route of administration.

[0071] The kidneys and liver are the major organs for the elimination ofrFGF-2. In particular, the kidneys have a protein cutoff of about 60 kDand thus retain serum albumin (MW 60 kD). However, the FGF-2 of SEQ IDNO: 5 has a molecular weight of about 16 kD. Accordingly, renalexcretion is to be expected. In a radiolabelled biodistribution study ofcommercially available bovine FGF-2 (bFGF-2), both the liver and thekidney were shown to contain high counts of the radiolabelled bFGF-2 at1 hour after IV or IC injection. In the same study, FGF-2 appeared tobind to red blood cells, however these results were not confirmed by invitro analysis of the whole blood. In a published study, wherein anotherrecombinant iodinated form of bFGF-2 was given to rats, the liver wasidentified as the major organ of elimination. Whalen et al., “The Fateof Intravenously Administered bFGF and the Effect of Heparin,” GrowthFactors, 1:157-164 (1989). More particularly, it is known that FGF-2binds in the general circulation to (α₂-macroglobulin and that thiscomplex is internalized by receptors on the Kupffer cells. Whalen et al.(1989) and LaMarre et al., “Cytokine Binding and Clearance Properties ofProteinase-Activated Alpha-2-Macroglobulins,” Lab. Invest., 65:3-14(1991). Labelled FGF-2 fragments were not found in the plasma, but theywere found in the urine and corresponded in size to intracellularbreakdown products. When FGF-2 was administered in combination withheparin, the renal excretion of FGF-2 was increased. Whalen et al.(1989). The FGF-2 molecule, which is cationic when not complexed withheparin, is likely repelled by the cationic heparin sulfate of theglomerular basement membrane. The FGF-2/heparin complex is moreneutrally charged, and therefore is more easily filtered and excreted bythe kidney.

[0072] We determined the pharmacokinetics of rFGF-2(SEQ ID NO: 5) afterintravenous (IV) and intracoronary (IC) administration in domesticYorkshire pigs, after IV dosing in Sprague Dawley (“SD”) rats, and afterIC administration in CAD human patients. In all species, the rFGF-2plasma concentrations after IV and/or IC injection followed abiexponential curve with an initial steep slope and considerabledecrease over several log scales (the distribution phase) during thefirst hour, followed by a more moderate decline (the elimination phase).FIG. 1 provides a plasma concentration versus time curve showing thesephases in humans after IC administration of rFGF-2 of SEQ ID NO: 5 as afunction of the following doses: 0.33 μg/kg, 0.65 μg/kg, 2 μg/kg, 6μg/kg, 12 μg/kg, and 24 μg/kg of lean body mass (LBM). The plasmaconcentrations of rFGF-2 of SEQ ID NO: 5 were determined by acommercially available ELISA (R &D Systems, Minneapolis, Minn.) that wasmarketed for analysis of human FGF-2. The ELISA assay showed 100%cross-reactivity with the rFGF-2 of SEQ ID NO: 5. Other members of theFGF family, as well as many other cytokines, were not detected by thisassay. Further, heparin does not interfere with the assay.

[0073] The design of these pharmokinetic studies, pharmacokineticparameters, and conclusions are listed in Tables 5 and 6 for studies inpigs and rats, respectively. The reader is referred to these tables forthe specific details. However, among the points to be noted are that thehalf-life (Ty,) was 2.8±0.8 to 3.5 hours following a single IC infusionfor the single component model for animals having a clearance (CL) of702±311 to 609±350 ml/hr/kg. The results of this study show that thepharmacokinetics of the recombinant bFGF-2 of SEQ ID NO: 5 weresubstantially identical regardless of whether the animals were dosed viathe IC or IV routes. See Table 5. In pigs, the maximum tolerated dose ofrecombinant bFGF-2 was 6.5 μg/kg. Among the other pharinacokineticresults to be taken from Tables 5 and 6 of these studies is that thereis a fast distribution phase followed by a more moderate eliminationphase, and dose linearity as reported in FIG. 1 for humans. Also, therewere no gender differences. Further, the three compartment model wasanalyzed for pigs receiving 70 U/kg of heparin approximately (“˜”) 15minutes before receiving 0.65-6.5 μg/kg by 5-10 minute IC infusion. Thehalf lives (T_(½α) T_(½β) and T_(½γ) ) for the three compartments were1.5 minutes, 17 minutes, and 6.6 hours, respectively. In these animals,the initial volume (“V₁”) was approximately the plasma volume, and thesteady state volume (“V_(SS”) was approximately) 10-fold the plasmavolume. See Table 5. In pigs, the binding of recombinant bFGF-2 of SEQID NO: 5 to circulating heparin appears to decrease biodistribution andelimination. Likewise, in rats, both the volume of TABLE 5Pharmacokinetics (PK) and Pharmacodynamics of rFGF-2 in Pigs AnimalsDosing Regimen PK Parameters Results Domestic Yorkshire pigs   2-20μg/kg IV bolus CL = 702 ± 311 mL/hr/kg Systemic PK identical between IVand IC route under general anesthesia   2-20 μg/kg IC bolus T½ = 2.8 ±0.8 hr. Fast distribution phase (n = 13; 30 ± 5 kg)  20 μg/kg by 10-minIC Dose-linearity infusion Transient decreases of MAP  70 U/kg heparin ˜15 min before rFGF-2 Domestic Yorkshire pigs 0.65-6.5 μg/kg by 5-min CL= 609 ± 350 ml/hr/kg No gender difference in PK under general anesthesiaIC infusion T½ = ˜3.5 hr Biphasic decline of plasma rFGF-2 (n = 17; 26 ±4 kg)  70 U/kg heparin ˜ 15 3-Comp. Model; Dose-linearity min beforerFGF-2 T½α = 1.5 min V₁ equal to ˜ plasma volume T½β = 17 min V₅₅ equalto ˜ 10-fold volume T½γ = 6.6 hr Magnitude and duration of MAP decreasecorrelated CL = 580 ml/hr/kg with rFGF-2 dose and peak plasma level V₁ =55 ml/kg V₅₅ = 523 ml/kg Domestic Yorkshire pigs 6.5 μg/kg weekly by 5Without Heparin The rFGF-2 distribution phase was less steep, the undergeneral anesthesia min IV infusion for 6 (Doses 1-6): volume ofdistribution smaller, and clearance was (n = 6; 25 ± 5 kg) weeks T½ =2-6 hr slower with heparin-pretreatment  70 U/kg heparin 10 min CL =777-2749 ml/hr/kg Binding of rFGF-2 to circulating heparin appears tobefore rFGF-2 (n = 3), or V₅₅ = 871-12,500 ml/kg decreasebiodistribution and elimination rFGF-2 alone (n = 3) With Heparin Bothvolume and clearance of rFGF-2 increased at (Doses 1-6): later doses(potential receptor upregulation), but more T½ = 2-3 hr so in theabsence of heparin CL = 235-347 ml/hr/kg Magnitude and duration of MAPdecreases were V₅₅ = 71-153 ml/kg similar with or without heparin

[0074] distribution and the clearance of rFGF-2 were smaller whenheparin was administered. See Table 6. Further, the greatest and mostfavorable changes on clearance of FGF-2 were found when heparin wasadministered within ±15 minutes, preferably immediately prior to rFGF-2IC infusion. See Table 6.

[0075] The pharmacokinetics of the rFGF-2 of SEQ ID NO: 5 was studied inhumans, diagnosed with CAD despite optimal medical management, in aPhase 1 clinical study supporting this filing. The doses of rFGF-2employed in that Phase 1 study were 0.33 μg/kg, 0.65 μg/kg, 2 μg/kg, 6μg/kg, 12 μg/kg, and 24 μg/kg of lean body mass (LBM), and all doseswere administered by a 20 minute IC infusion (10 minutes into each oftwo patent coronary vessels) after pretreating the patient with 40 U/kgheparin which was administered IV or IC 1-95 minutes before rFGF-2infusion. FIGS. 1-3 herein summarize the data underlying those results.In particular, FIG. 1 is a plot of the mean rFGF-2 plasma concentrationversus time (hours) for the six different doses of rFGF-2 (SEQ ID NO: 5)administered by IC infusion as described above over a 20 minute period.FIG. 1 shows dose linearity and a biphasic plasma level decline, i.e., afast distribution phase during the first hour, followed by anelimination phase with T_(½) of 1.9±2.2 hours. The dose linearity ismore readily seen in FIG. 2, which is a plot of the individual patientrFGF-2 area under the curve (AUC) in pg·hr/ml for FIG. 1 for each of thesix doses of rFGF-2 administered. FIG. 3 is a plot individual humanpatient rFGF-2 dose normalized AUCs versus time of heparin dose in“minutes prior to rFGF-2 infusion” and shows the influence of timing ofheparin administration on rFGF-2 AUC. FIG. 3 shows that the greatestAUC/dose was achieved when an effective amount of a glycosoaminoglycan,such as heparin, was preadministered within 30 minutes or less of ICrFGF-2 infusion, more preferably within 20 minutes or less of IC rFGF-2infusion. Typically, an effective amount of a glycosoaminoglycan is40-70 U/kg heparin. These pharmacokinetic results are summarized inTable 7 herein.

[0076] The rFGF-2 distribution phase was less steep with heparin, thevolume of distribution smaller, and the clearance slower, as compared torFGF-2 without heparin. It appears that the complex of rFGF-2 withcirculating heparin decreases the biodistribution and elimination ofrFGF-2. Although the binding of FGF-2 to heparin-like structures isstrong (dissociation constant ˜2×10⁻⁹ M), the binding of FGF-2 to theTABLE 6 Pharmacokinetics (PK) of rFGF-2 in Rats Animals Dosing RegimenPK Parameters Results Conscious SD rats  3-100 μg/kg bolus IV T½ = 1.1 ±0.51 hr Fast distribution phase (n = 18; 322 ± 93 g) injection CL = 4480± 2700 ml/hr/kg Apparent dose-linearity V₅₅ = 1924 ± 1254 ml/kgconscious SD rats 30-300 μg/kg weekly by T½ = 1.4 ± 0.13 hrTime-invariant PK; plasma profiles, PK parameters and (n = 54; 149 ± 12g) bolus IV injection for 6 CL = 1691 ± 169 ml/hr/kg AUCs were similarover time weeks V₅₅ = 1942 ± 358 ml/kg Dose-linearity No heparinpretreatment Conscious SD rats Time-Averaged PK In all cases, heparinincreased the rFGF-2 plasma levels (27 males; 381 ± 48 g; Parameters:Both volume of distribution and clearance of 20 females; 268 ± 22 g) T½CL V₅₅ rFGF-2 were smaller with heparin hr. ml/hr/kg ml/kg Greatestchanges on CL and V₅₅ were observed when 30 μg/kg bolus IV 0.75 43322389 heparin was administered immediately prior to rFGF-2 injection Noheparin 40 U/kg IV Heparin: at ˜ 15 min 0.91 1728  844 just prior torFGF-2 1.3  516  147 at +15 min 1.2 1158  626 at +3 hr 0.93 1338 1351

[0077] TABLE 7 Pharmacokinetics of rFGF-2 in Human Subjects DosingRegimen PK Parameters Results Patients with CAD 0.33-24 μg/kg LBM*Preliminary PK data Biphasic plasma level decline: fast distribution by20-min IC injection from 0.33-24 μg/kg phase during 1 hr; followed byelimination phase with (10 min in left main doses (n = 32) T½approximately (˜) 2 hr coronary artery + 10 T½ = 1.9 ± 2.2 hrDose-linearity min in right main CL = 264 ± 150 ml/hr/kg Greater rFGF-2exposure (as measured by the coronary artery) V₅₅ = 184 ± 74 ml/kg AUC)was found when heparin pretreatment was 0.33 μg/kg, n = 4 given closerto the start of the rFGF-2 infusion, 0.65 μg/kg, n = 4 preferably within20 minutes   2 μg/kg, n = 8   6 μg/kg, n = 4   12 μg/kg, n = 4   24μg/kg, n = 8   36 μg/kg, n = 10   48 μg/kg, n = 10   40 U/kg heparinpretreatment, 1-95 min before rFGF-2 infusion

[0078] FGF-2 receptor is approximately two orders of magnitude higher(dissociation constant ˜2×10⁻¹¹ M). Moscatelli et al., (1991).

[0079] In addition, the complexation of the rFGF-2 of SEQ ID NO: 5 witha glycosoaminoglycan, such as a heparin, might increase signaltransduction and mitogenesis, and/or protect the rFGF-2 from enzymaticdegradation.

[0080] The examples, which follow, provide more details on the selectioncriterion and the Phase I clinical trial that gave rise to the datadiscussed above.

EXAMPLE 1 “Unit Dose of rFGF-2 Employed in the Phase I Clinical Trial”

[0081] The rFGF-2 of SEQ ID NO: 5 was formulated as a unit dose andpharmaceutical composition and administered to rats, pigs, andultimately to humans in the Phase I clinical trial referenced herein.The various formulations are described below.

[0082] The rFGF-2 Unit Dose was provided as a liquid in 3 cc type Iglass vials with a laminated gray butyl rubber stopper and red flip-offoverseal. The rFGF- 2 unit dose contained 1.2 ml of 0.3 mg/ml rFGF-2 ofSEQ ID NO: 5 in 10 mM sodium citrate, 10 mM monothioglycerol, 1 mMdisodium dihydrate EDTA (molecular weight 372.2), 135 mM sodiumchloride, pH 5.0. Thus, in absolute terms, each vial (and unit dose)contained 0.36 mg rFGF-2. The vials containing the unit dose in liquidform were stored at 2° to 8° C.

[0083] The rFGF Diluent was supplied in 5 cc type I glass vials with alaminated gray butyl rubber stopper and red flip-off overseal. TherFGF-2 diluent contains 10 mM sodium citrate, 10 mM monothioglycerol,135 mM sodium chloride, pH 5.0. Each vial contained 5.2 ml of rFGF-2diluent solution that was stored at 2° to 8° C.

[0084] The rFGF-2 Pharmaceutical Composition that was infused wasprepared by diluting the rFGF-2 unit dose with the rFGF diluent suchthat the infusion volume is 10 ml. In order to keep the EDTAconcentration below the limit of 100 μg/ml, the total infuision volumewas increased to 20 ml when proportionately higher absolute amounts ofFGF-2 were administered to patients with high body weights.

EXAMPLE 2 “Selection Criteria for Patients with Coronary Artery Diseasefor Treatment with rFGF-2”

[0085] The following selection criteria were applied to Phase I patientswith coronary artery disease, whose activities were limited by coronaryischemia despite optimal medical management, and who were not candidatesfor approved revascularization therapies:

Inclusion criteria: Subject is eligible if:

[0086] Male or female, greater than or equal to 18 years of age

[0087] Diagnosis of coronary artery disease (CAD)

[0088] Suboptimal candidates for approved revascularization procedures,e.g., angioplasty, stents, coronary artery bypass graft (CABG) (orrefuses those interventions)

[0089] Able to exercise at least three minutes using a modified Bruceprotocol and limited by coronary ischemia

[0090] Inducible and reversible defect of at least 20% myocardium onpharmacologically stressed thallium sestamibi scan

[0091] CBC, platelets, serum chemistry within clinically acceptablerange for required cardiac catheterization

[0092] Normal INR, or if anticoagulated with Coumadin, INR <2.0

[0093] Willing and able to give written informed consent to participatein this study, including all required study procedures and follow-upvisits

Exclusion criteria: Subject is not eligible if:

[0094] Malignancy: any history of malignancy within past ten years, withthe exception of curatively treated basal cell carcinoma

[0095] Ocular conditions: proliferative retinopathy, severenon-proliferative retinopathy, retinal vein occlusion, Eales' disease,or macular edema or funduscopy by ophthalmologist: history ofintraocular surgery within six months

[0096] Renal function: creatinine clearance below normal range adjustedfor age; protein >250 mg or microalbumin >30 mg/24 h urine

[0097] Class IV heart failure (New York Heart Association)

[0098] Ejection fraction <20% by echocardiogram, thallium scan, MRI orgated pooled blood scan (MUGA)

[0099] Hemodynamically relevant arrhythmias (e.g., ventricularfibrillation, sustained ventricular tachycardia)

[0100] Severe valvular stenosis (aortic area <1.0 cm², mitral area <1.2cm²), or severe valvular insufficiency

[0101] Marked increase in angina or unstable angina within three weeks

[0102] History of myocardial infarction (MI) within three months

[0103] History of transient ischemic attack (TIA) or stroke within sixmonths

[0104] History of CABG, angioplasty or stent within six months

[0105] History of treatment with transmyocardial laserrevascularization, rFGF-2, or vascular enodothelial growth factor (VEGF)within six months

[0106] Females of child-bearing potential or nursing mothers

[0107] Any pathological fibrosis, e.g., pulmonary fibrosis, scleroderma

[0108] Known vascular malformation, e.g., AV malformation, hemangiomas

[0109] Coexistence of any disease which might interfere with assessmentof symptoms of CAD, e.g. pericarditis, costochondritis, esophagitis,systemic vasculitis, sickle cell disease

[0110] Coexistence of any disease which limits performance of modifiedBruce protocol exercise stress test, e.g., paralysis or amputation of alower extremity, severe arthritis or lower extremities, severe chronicobstructive pulmonary disease (COPD)

[0111] Participation in clinical trials of investigational agents,devices or procedures within thirty days (or scheduled within sixty daysof study drug)

[0112] Known hypersensitivity to rFGF-2 or related compounds

[0113] Any condition which makes the subject unsuitable forparticipation in this study in the opinion of the investigator, e.g.,psychosis, severe mental retardation, inability to communicate withstudy personnel, drug or alcohol abuse

EXAMPLE 3 “Phase I Clinical Study on Recombinant FGF-2 (SEQ ID NO: 1)Administered to Humans”

[0114] Recombinant bFGF-2 of SEQ ID NO: 5 was administered to 52 humanpatients with severe CAD, who remained symptomatic despite optimalmedical management and who refused or were suboptimal candidates forsurgical or percutaneous revascularization, in a Phase I open label,single administration, dose escalation, two-site trial. The drug wasadministered as a single 20 minute infusion divided between two majorsources of coronary blood supply (IC), using standard techniques forpositioning a catheter into the patient's coronary artery (such asalready employed in angioplasty). The doses (μg/kg) of rFGF-2administered were 0.33 (n=4), 0.65 (n=4), 2.0 (n=8), 6.0 (n=4), 12.0(n=4), 24 (n=8), 36 (n=10), and 48 (n=10) of rFGF-2 of SEQ ID NO: 5.Angina frequency and quality of life was assessed by the Seattle AnginaQuestionnaire (SAQ) at a baseline (before rFGF-2 administration) and atabout 60 days after rbFGF-2 administration. Exercise tolerance time(ETT) was assessed by the threadmill test. Rest/exercise nuclearperfusion and gated sestamibi-determined rest ejection fraction (EF),and magnetic resonance imaging (MRI) were assessed at baseline, and at30 days and 60 days post FGF-2 administration. Other end points thatwere evaluated included MRI (to objectively measure ejection fraction(EF), normal wall motion (NWM), targeted wall motion (TWM), normal wallthickness (NWT), targeted wall thickness (TWT), ischemic area zone andcollateral extent). See Tables 2-4, respectively.

[0115] The preliminary safety results indicate that serious events werenot dose related. Thus far, of the eight dosage groups, there were threedeaths in the lowest dosage groups, i.e., at 0.65 μg/kg (Day 23), at 2.0μg/kg (Day 57), and at 6.0 μg/kg (Day 63). There were sixhospitalizations for acute myocardial infarction (MI) in three patients,i.e., one patient from each of groups 1 (0.33 μg/kg), 3 (2.0 μg/kg), and4 (6.0 μg/kg). One of the three patients accounted for four of the sixhospitalizations for acute MI. There was also one large B cell lymphomathat was diagnosed three weeks after dosing in a patient in group 4. Thepatient died at two months post dosing. Acute hypotension, seen athigher doses during or just subsequent to infusion, was managed byadministration of fluids without need for a vasopressor. The maximumtolerated dose of rFGF-2 (SEQ ID NO: 2) was defined as 36 μg/kg. Dosesof rFGF-2 up to 48 μg/kg IC were managed in patients with aggressivefluid management. However, they were not tolerated due to acute and/ororthostatic hypotension in two out of ten patients. The half-life inhumans of the IC infused rFGF-2 was about one hour.

[0116] The human patients in this study that were treated with a singleIC infusion of rFGF-2 of SEQ ID NO: 5 exhibited a mean increase in ETTof 1.5 to 2 minutes. This is especially significant because an increasein ETT of >30 seconds is considered significant and a benchmark forevaluating alternative therapies, such as angioplasty. The anginafrequency and quality of life, as measured by SAQ, showed a significantimprovement at 57 days in all five subscales for the 28 patients (n=28)tested. See Tables 2 and 3. In particular, the mean changes in scoresfor the five criteria evaluated by the SAQ ranged from 13 to 36 with amean change of 8 or more considered “clinically significant.” See Table2.

[0117] Magnetic resonance imaging (MRI) showed objective improvementsfollowing administration of a single unit dose of the bFGF-2 of SEQ IDNO: 5, including increased targeted wall motion at 30 and 60 days(p<0.05), and increased targeted wall thickening at 60 days (p<0.01).MRI further showed improved regional wall motion, and increasedmyocardial perfusion and collateral development in the targeted area forboth the lower dose (0.33 μg/kg and 0.65 μg/kg) and higher dose (2.0μg/kg and 12.0 μg/kg) groups in an 11 patient test group (n=11).

[0118] Abnormal perfusion zone, which was assessed at one of the siteson 28 patients, decreased significantly at 30 and 60 days (p<0.001).

[0119] In addition to the above criterion (ie., ETT SAQ, MRI), atreatment is considered very successful if the angiogenic effects lastat least six months. In the present Phase I study, the unexpectedlysuperior angiogenic effects were observed to last for 57-60 days in alldosage groups. (See Tables 2-4.) Based upon the results alreadyobtained, it is expected that the angiogenic effects would last twelvemonths or more but at least six months, at which time the procedurecould be repeated, if necessary.

EXAMPLE 4 “Proposed Phase II Clinical Study On Recombinant FGF-2 (SEQ IDNO: 1) Administered to Humans to Treat Coronary Artery Disease”

[0120] The Phase II clinical trial of rFGF-2 for treating human patientsfor coronary artery disease is performed as a double blind/placebocontrolled study having four arms: placebo, 0.3 μg/kg, 3 μg/kg, and 30μg/kg administered IC.

EXAMPLE 5 “Unit Dose and Pharmaceutical Composition of rFGF-2 for thePhase II Human Clinical Trial”

[0121] The rFGF-2 of SEQ ID NO: 5 was formulated as a unit dose andpharmaceutical composition for administration to humans in the Phase IIclinical trial referenced herein. The various formulations are describedbelow. The rFGF-2 Unit Dose was prepared as a liquid in 5 cc type Iglass vials with a laminated gray butyl rubber stopper and red flip-offoverseal. The rFGF-2 formulation contains 0.3 mg/ml rFGF-2 of SEQ ID NO:5 in 10 mM sodium citrate, 10 mM monothioglycerol, 0.3 mM disodiumdihydrate EDTA (molecular weight 372.2), 135 mM sodium chloride, pH 5.0.Each vial contained 3.7 ml of rFGF-2 drug product solution (1.11 mgrFGF-2 per vial). The resulting unit dose in liquid form is stored atless than −60° C. The above described unit dose is diluted with the“rFGF-2 placebo.” Depending on the size of the patient, the contents ofseveral of the vials may be confirmed to produce a unit dose of 36 mg/kgfor the Phase II study.

[0122] The rFGF Placebo is supplied as a clear colorless liquid in 5 cctype I glass vials with a laminated gray butyl rubber stopper and redflip-off overseal. The rFGF-2 placebo is indistinguishable in appearancefrom the drug product and has the following formulation: 10 mM sodiumcitrate, 10 mM monothioglycerol, 0.3 mM disodium dihydrate EDTA(molecular weight 372.2), 135 mM sodium chloride, pH 5.0. Each vialcontains 5.2 ml of rFGF-2 placebo solution. Like the unit dose, therFGF-2 placebo is stored at 2° to 8° C.

[0123] The rFGF-2 Pharmaceutical Composition that is infused is preparedby diluting the rFGF-2 unit dose with the rFGF diluent such that theinfusion volume is 20 ml for Phase II.

EXAMPLE 6 “Selection Criteria for CAD Patients for the Phase II HumanClinical Trial of IC rFGF-2”

[0124] Accordingly, the above described evidence of an unexpectedlysuperior improvement in quality of life and of increased angiogenicefficacy in humans who were administered a single unit dosage of rFGF-2in accordance with this invention, supports the patentability of theApplicants' unit dose, pharmaceutical composition and method of usingthe same.

[0125] All publications and patent applications mentioned in thespecification are indicative of the level of those skilled in the art towhich this invention pertains. All publications and patent applicationsare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference in its entirety.

[0126] Those skilled in the art will recognize, or be able to ascertainusing no more than routine experimentation, many equivalents to thespecific embodiments of the invention described herein. Such equivalentsare intended to be encompassed by the present invention describedherein.

1 15 1 140 PRT Human FGF-1 1 Phe Asn Leu Pro Pro Gly Asn Tyr Lys Lys ProLys Leu Leu Tyr Cys 1 5 10 15 Ser Asn Gly Gly His Phe Leu Arg Ile LeuPro Asp Gly Thr Tyr Asp 20 25 30 Gly Thr Arg Asp Arg Ser Asp Gln His IleGln Leu Gln Leu Ser Ala 35 40 45 Glu Ser Tyr Gly Glu Tyr Tyr Ile Lys SerThr Glu Thr Gly Gln Tyr 50 55 60 Leu Ala Met Asp Thr Asp Gly Leu Leu TyrGly Ser Gln Thr Pro Asn 65 70 75 80 Glu Glu Cys Leu Phe Leu Glu Arg LeuGlu Glu Asn His Tyr Asn Thr 85 90 95 Tyr Ile Ser Lys Lys His Ala Glu LysAsn Trp Phe Tyr Gly Leu Lys 100 105 110 Lys Asn Gly Ser Cys Lys Arg GlyPro Arg Thr His Tyr Gly Gln Lys 115 120 125 Ala Ile Leu Phe Leu Pro LeuPro Tyr Ser Ser Asp 130 135 140 2 140 PRT bovine FGF-1 2 Phe Asn Leu ProLeu Gly Asn Tyr Lys Lys Pro Lys Leu Leu Tyr Cys 1 5 10 15 Ser Asn GlyGly Tyr Phe Leu Arg Ile Leu Pro Asp Gly Thr Val Asp 20 25 30 Gly Thr LysAsp Arg Ser Asp Gly His Ile Gln Leu Phe Leu Cys Ala 35 40 45 Glu Ser IleGly Glu Val Tyr Ile Lys Ser Thr Glu Thr Gly Gln Phe 50 55 60 Leu Ala MetAsp Thr Asp Gly Leu Leu Tyr Gly Ser Gln Thr Pro Asp 65 70 75 80 Glu GluCys Leu Phe Leu Glu Arg Leu Glu Glu Asn His Tyr Asn Thr 85 90 95 Tyr IleSer Lys Lys His Ala Glu Lys His Trp Phe Val Gly Leu Lys 100 105 110 LysAsn Gly Arg Ser Lys Leu Glu Pro Arg Thr His Phe Gly Gln Lys 115 120 125Ala Ile Leu Phe Leu Pro Leu Pro Val Ser Ser Asp 130 135 140 3 146 PRTHuman FGF-2 3 Pro Ala Leu Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro ProGly His 1 5 10 15 Phe Lys Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly GlyPhe Phe Leu 20 25 30 Arg Ile His Pro Asp Gly Arg Val Asp Gly Val Arg GluLys Ser Asp 35 40 45 Pro His Ile Lys Leu Gln Leu Gln Ala Glu Glu Arg GlyVal Val Ser 50 55 60 Ile Lys Gly Val Cys Ala Asn Arg Tyr Leu Ala Met LysGlu Asp Gly 65 70 75 80 Arg Leu Leu Ala Ser Lys Cys Val Thr Asp Glu CysPhe Phe Phe Glu 85 90 95 Arg Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg SerArg Lys Tyr Thr 100 105 110 Ser Trp Tyr Val Ala Leu Lys Arg Thr Gly GlnTyr Lys Leu Gly Ser 115 120 125 Lys Thr Gly Pro Gly Gln Lys Ala Ile LeuPhe Leu Pro Met Ser Ala 130 135 140 Lys Ser 145 4 442 DNA bovine FGF-2CDS (1)..(438) 4 cca gcc cta cca gaa gat ggg ggg tcc ggg gcc ttc cca ccaggg cac 48 Pro Ala Leu Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro GlyHis 1 5 10 15 ttc aaa gat cca aaa cga cta tat tgt aaa aac ggg ggg ttcttc cta 96 Phe Lys Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly Phe PheLeu 20 25 30 cga atc cac cca gat ggg cga gta gat ggg gta cga gaa aaa tccgat 144 Arg Ile His Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp35 40 45 cca cac atc aaa cta caa cta caa gcc gaa gaa cga ggg gta gta tcc192 Pro His Ile Lys Leu Gln Leu Gln Ala Glu Glu Arg Gly Val Val Ser 5055 60 atc aaa ggg gta tgt gcc aac cga tat cta gcc atg aaa gaa gat ggg240 Ile Lys Gly Val Cys Ala Asn Arg Tyr Leu Ala Met Lys Glu Asp Gly 6570 75 80 cga cta cta gcc tcc aaa tgt gta acc gat gaa tgt ttc ttc ttc gaa288 Arg Leu Leu Ala Ser Lys Cys Val Thr Asp Glu Cys Phe Phe Phe Glu 8590 95 cga cta gaa tcc aac aac tat aac acc tat cga tcc cga aaa tat tcc336 Arg Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Arg Lys Tyr Ser 100105 110 tcc tgg tat gta gcc cta aaa cga acc ggg caa tat aaa cta ggg cca384 Ser Trp Tyr Val Ala Leu Lys Arg Thr Gly Gln Tyr Lys Leu Gly Pro 115120 125 aaa acc ggg cca ggg caa aaa gcc atc cta ttc cta cca atg tcc gcc432 Lys Thr Gly Pro Gly Gln Lys Ala Ile Leu Phe Leu Pro Met Ser Ala 130135 140 aaa tcc taag 442 Lys Ser 145 5 146 PRT bovine FGF-2 5 Pro AlaLeu Pro Glu Asp Gly Gly Ser Gly Ala Phe Pro Pro Gly His 1 5 10 15 PheLys Asp Pro Lys Arg Leu Tyr Cys Lys Asn Gly Gly Phe Phe Leu 20 25 30 ArgIle His Pro Asp Gly Arg Val Asp Gly Val Arg Glu Lys Ser Asp 35 40 45 ProHis Ile Lys Leu Gln Leu Gln Ala Glu Glu Arg Gly Val Val Ser 50 55 60 IleLys Gly Val Cys Ala Asn Arg Tyr Leu Ala Met Lys Glu Asp Gly 65 70 75 80Arg Leu Leu Ala Ser Lys Cys Val Thr Asp Glu Cys Phe Phe Phe Glu 85 90 95Arg Leu Glu Ser Asn Asn Tyr Asn Thr Tyr Arg Ser Arg Lys Tyr Ser 100 105110 Ser Trp Tyr Val Ala Leu Lys Arg Thr Gly Gln Tyr Lys Leu Gly Pro 115120 125 Lys Thr Gly Pro Gly Gln Lys Ala Ile Leu Phe Leu Pro Met Ser Ala130 135 140 Lys Ser 145 6 9 PRT Bovis bovinus 6 Met Ala Ala Gly Ser IleThr Thr Leu 1 5 7 240 PRT Murine FGF-3 7 Met Gly Leu Ile Trp Leu Leu LeuLeu Ser Leu Leu Glu Pro Ser Trp 1 5 10 15 Pro Thr Thr Gly Pro Gly ThrArg Leu Arg Arg Asp Ala Gly Gly Arg 20 25 30 Gly Gly Val Tyr Glu His LeuGly Gly Ala Pro Arg Arg Arg Lys Leu 35 40 45 Tyr Cys Ala Thr Lys Tyr HisLeu Gln Leu His Pro Ser Gly Arg Val 50 55 60 Asn Gly Ser Leu Glu Asn SerAla Tyr Ser Ile Leu Glu Ile Thr Ala 65 70 75 80 Val Glu Val Gly Val ValAla Ile Lys Gly Leu Phe Ser Gly Arg Tyr 85 90 95 Leu Ala Met Asn Lys ArgGly Arg Leu Tyr Ala Ser Asp His Tyr Asn 100 105 110 Ala Glu Cys Glu PheVal Glu Arg Ile His Glu Leu Gly Tyr Asn Thr 115 120 125 Tyr Ala Ser ArgLeu Tyr Arg Thr Gly Ser Ser Gly Pro Gly Ala Gln 130 135 140 Arg Gln ProGly Ala Gln Arg Pro Trp Tyr Val Ser Val Asn Gly Lys 145 150 155 160 GlyArg Pro Arg Arg Gly Phe Lys Thr Arg Arg Thr Gln Lys Ser Ser 165 170 175Leu Phe Leu Pro Arg Val Leu Gly His Lys Asp His Glu Met Val Arg 180 185190 Leu Leu Gln Ser Ser Gln Pro Arg Ala Pro Gly Glu Gly Ser Gln Pro 195200 205 Arg Gln Arg Arg Gln Lys Lys Gln Ser Pro Gly Asp His Gly Lys Met210 215 220 Glu Thr Leu Ser Thr Arg Ala Thr Pro Ser Thr Gln Leu His ThrGly 225 230 235 240 8 205 PRT Human FGF-4 8 Ser Gly Pro Gly Thr Ala AlaVal Ala Leu Leu Pro Ala Val Leu Leu 1 5 10 15 Ala Leu Leu Ala Pro TrpAla Gly Arg Gly Gly Ala Ala Ala Pro Thr 20 25 30 Ala Pro Asn Gly Thr LeuGlu Ala Glu Leu Glu Arg Arg Trp Glu Ser 35 40 45 Leu Val Ala Leu Ser LeuAla Arg Leu Pro Val Ala Ala Gln Pro Lys 50 55 60 Glu Ala Ala Val Gln SerGly Ala Gly Asp Tyr Leu Leu Gly Ile Lys 65 70 75 80 Arg Leu Arg Arg LeuTyr Cys Asn Val Gly Ile Gly Phe His Leu Gln 85 90 95 Ala Leu Pro Asp GlyArg Ile Gly Gly Ala His Ala Asp Thr Arg Asp 100 105 110 Ser Leu Leu GluLeu Ser Pro Val Glu Arg Gly Val Val Ser Ile Phe 115 120 125 Gly Val AlaSer Arg Phe Phe Val Ala Met Ser Ser Lys Gly Lys Leu 130 135 140 Tyr GlySer Pro Phe Phe Thr Asp Glu Cys Thr Phe Lys Glu Ile Leu 145 150 155 160Leu Pro Asn Asn Tyr Asn Ala Tyr Glu Ser Tyr Lys Tyr Pro Gly Met 165 170175 Phe Ile Ala Leu Ser Lys Asn Gly Lys Thr Lys Lys Gly Asn Arg Val 180185 190 Ser Pro Thr Met Lys Val Thr His Phe Leu Pro Arg Leu 195 200 2059 266 PRT Human FGF-5 9 Ser Leu Ser Phe Leu Leu Leu Leu Phe Phe Ser HisLeu Ile Leu Ser 1 5 10 15 Ala Trp Ala His Gly Glu Lys Arg Leu Ala ProLys Gly Gln Pro Gly 20 25 30 Pro Ala Ala Thr Asp Arg Asn Pro Arg Gly SerSer Ser Arg Gln Ser 35 40 45 Ser Ser Ser Ala Met Ser Ser Ser Ser Ala SerSer Ser Pro Ala Ala 50 55 60 Ser Leu Gly Ser Gln Gly Ser Gly Leu Glu GlnSer Ser Phe Gln Trp 65 70 75 80 Ser Leu Gly Ala Arg Thr Gly Ser Leu TyrCys Arg Val Gly Ile Gly 85 90 95 Phe His Leu Gln Ile Tyr Pro Asp Gly LysVal Asn Gly Ser His Glu 100 105 110 Ala Asn Met Leu Ser Val Leu Glu IlePhe Ala Val Ser Gln Gly Ile 115 120 125 Val Gly Ile Arg Gly Val Phe SerAsn Lys Phe Leu Ala Met Ser Lys 130 135 140 Lys Gly Lys Leu His Ala SerAla Lys Phe Thr Asp Asp Cys Lys Phe 145 150 155 160 Arg Glu Arg Phe GlnGlu Asn Ser Tyr Asn Thr Tyr Ala Ser Ala Ile 165 170 175 His Arg Thr GluLys Thr Gly Arg Glu Trp Tyr Val Ala Leu Asn Lys 180 185 190 Arg Gly LysAla Lys Arg Gly Cys Ser Pro Arg Val Lys Pro Gln His 195 200 205 Ile SerThr His Phe Leu Pro Arg Phe Lys Gln Ser Glu Gln Pro Glu 210 215 220 LeuSer Phe Thr Val Thr Val Pro Glu Lys Lys Lys Pro Pro Ser Pro 225 230 235240 Ile Lys Pro Lys Ile Pro Leu Ser Ala Pro Arg Lys Asn Thr Asn Ser 245250 255 Val Lys Tyr Arg Leu Lys Phe Arg Phe Gly 260 265 10 207 PRT HumanFGF-6 10 Ala Leu Gly Gln Lys Leu Phe Ile Thr Met Ser Arg Gly Ala Gly Arg1 5 10 15 Leu Gln Gly Thr Leu Trp Ala Leu Val Phe Leu Gly Ile Leu ValGly 20 25 30 Met Val Val Pro Ser Pro Ala Gly Thr Arg Ala Asn Asn Thr LeuLeu 35 40 45 Asp Ser Arg Gly Trp Gly Thr Leu Leu Ser Arg Ser Arg Ala GlyLeu 50 55 60 Ala Gly Glu Ile Ala Gly Val Asn Trp Glu Ser Gly Tyr Leu ValGly 65 70 75 80 Ile Lys Arg Gln Arg Arg Leu Tyr Cys Asn Val Gly Ile GlyPhe His 85 90 95 Leu Gln Val Leu Pro Asp Gly Arg Ile Ser Gly Thr His GluGlu Asn 100 105 110 Pro Tyr Ser Leu Leu Glu Ile Ser Thr Val Glu Arg GlyVal Val Ser 115 120 125 Leu Phe Gly Val Arg Ser Ala Leu Phe Val Ala MetAsn Ser Lys Gly 130 135 140 Arg Leu Tyr Ala Thr Pro Ser Phe Gln Glu GluCys Lys Phe Arg Glu 145 150 155 160 Thr Leu Leu Pro Asn Asn Tyr Asn AlaTyr Glu Ser Asp Leu Tyr Gln 165 170 175 Gly Thr Tyr Ile Ala Leu Ser LysTyr Gly Arg Val Lys Arg Gly Ser 180 185 190 Lys Val Ser Pro Ile Met ThrVal Thr His Phe Leu Pro Arg Ile 195 200 205 11 193 PRT Human FGF-7 11Met His Lys Trp Ile Leu Thr Trp Ile Leu Pro Thr Leu Leu Tyr Arg 1 5 1015 Ser Cys Phe His Ile Ile Cys Leu Val Gly Thr Ile Ser Leu Ala Cys 20 2530 Asn Asp Met Thr Pro Glu Gln Met Ala Thr Asn Val Asn Cys Ser Ser 35 4045 Pro Glu Arg His Thr Arg Ser Tyr Asp Tyr Met Glu Gly Gly Asp Ile 50 5560 Arg Val Arg Arg Leu Phe Cys Arg Thr Gln Trp Tyr Leu Arg Ile Asp 65 7075 80 Lys Arg Gly Lys Val Lys Gly Thr Gln Glu Met Lys Asn Asn Tyr Asn 8590 95 Ile Met Glu Ile Arg Thr Val Ala Val Gly Ile Val Ala Ile Lys Gly100 105 110 Val Glu Ser Glu Phe Tyr Leu Ala Met Asn Lys Glu Gly Lys LeuTyr 115 120 125 Ala Lys Lys Glu Cys Asn Glu Asp Cys Asn Phe Lys Glu LeuIle Leu 130 135 140 Glu Asn His Tyr Asn Thr Tyr Ala Ser Ala Lys Trp ThrHis Asn Gly 145 150 155 160 Gly Glu Met Phe Val Ala Leu Asn Gln Lys GlyIle Pro Val Arg Gly 165 170 175 Lys Lys Thr Lys Lys Gln Lys Thr Ala HisPhe Leu Pro Met Ala Ile 180 185 190 Thr 12 215 PRT Murine FGF-8 12 MetGln Ser Pro Arg Ser Ala Leu Ser Cys Leu Leu Leu His Leu Leu 1 5 10 15Val Leu Cys Leu Gln Ala Gln Val Thr Val Gln Ser Ser Pro Asn Phe 20 25 30Thr Gln His Val Arg Glu Gln Ser Leu Val Thr Asp Gln Leu Ser Arg 35 40 45Arg Leu Ile Arg Thr Tyr Gln Leu Tyr Ser Arg Thr Ser Gly Lys His 50 55 60Val Gln Val Leu Ala Asn Lys Arg Ile Asn Ala Met Ala Phe Asp Gln 65 70 7580 Asp Pro Phe Ala Lys Leu Ile Val Glu Tyr Asp Thr Phe Gly Ser Arg 85 9095 Val Arg Val Arg Gly Ala Glu Thr Gly Leu Tyr Ile Cys Met Asn Lys 100105 110 Lys Gly Lys Leu Ile Ala Lys Ser Asn Gly Lys Gly Lys Asp Cys Val115 120 125 Phe Thr Phe Ile Val Ile Glu Asn Asn Tyr Thr Ala Leu Gln AsnAla 130 135 140 Lys Tyr Glu Gly Trp Tyr Met Ala Phe Thr Ala Lys Gly ArgPro Arg 145 150 155 160 Lys Gly Ser Lys Thr Arg Gln His Gln Arg Glu ValHis Phe Met Lys 165 170 175 Arg Leu Pro Arg Gly His His Thr Thr Glu GlnSer Leu Arg Phe Glu 180 185 190 Phe Leu Asn Tyr Pro Pro Phe Thr Arg SerLeu Arg Gly Ser Gln Arg 195 200 205 Thr Trp Ala Pro Glu Pro Arg 210 21513 208 PRT Human FGF-9 13 Met Ala Pro Leu Gly Glu Val Gly Asn Tyr PheGly Val Gln Asp Ala 1 5 10 15 Val Pro Phe Gly Asn Val Pro Val Leu ProVal Asp Ser Pro Val Leu 20 25 30 Leu Ser Asp His Leu Gly Gln Ser Glu AlaGly Gly Leu Pro Arg Gly 35 40 45 Pro Ala Val Thr Asp Leu Asp His Leu LysGly Ile Leu Arg Arg Arg 50 55 60 Gln Leu Tyr Cys Arg Thr Gly Phe His LeuGlu Ile Phe Pro Asn Gly 65 70 75 80 Thr Ile Gln Gly Thr Arg Lys Asp HisSer Arg Phe Gly Ile Leu Glu 85 90 95 Phe Ile Ser Ile Ala Val Gly Leu ValSer Ile Arg Gly Val Asp Ser 100 105 110 Gly Leu Tyr Leu Gly Met Asn GluLys Gly Glu Leu Tyr Gly Ser Glu 115 120 125 Lys Leu Thr Gln Glu Cys ValPhe Arg Glu Gln Phe Glu Glu Asn Trp 130 135 140 Tyr Asn Thr Tyr Ser SerAsn Leu Tyr Lys His Val Asp Thr Gly Arg 145 150 155 160 Arg Tyr Tyr ValAla Leu Asn Lys Asp Gly Thr Pro Arg Glu Gly Thr 165 170 175 Arg Thr LysArg His Gln Lys Phe Thr His Phe Leu Pro Arg Pro Val 180 185 190 Asp ProAsp Lys Val Pro Glu Leu Tyr Lys Asp Ile Leu Ser Gln Ser 195 200 205 14207 PRT Human FGF-98 14 Met Tyr Ser Ala Pro Ser Ala Cys Thr Cys Leu CysLeu His Phe Leu 1 5 10 15 Leu Leu Cys Phe Gln Val Gln Val Leu Val AlaGlu Glu Asn Val Asp 20 25 30 Phe Arg Ile His Val Glu Asn Gln Thr Arg AlaArg Asp Asp Val Ser 35 40 45 Arg Lys Gln Leu Arg Leu Tyr Gln Leu Tyr SerArg Thr Ser Gly Lys 50 55 60 His Ile Gln Val Leu Gly Arg Arg Ile Ser AlaArg Gly Glu Asp Gly 65 70 75 80 Asp Lys Tyr Ala Gln Leu Leu Val Glu ThrAsp Thr Phe Gly Ser Gln 85 90 95 Val Arg Ile Lys Gly Lys Glu Thr Glu PheTyr Leu Cys Met Asn Arg 100 105 110 Lys Gly Lys Leu Val Gly Lys Pro AspGly Thr Ser Lys Glu Cys Val 115 120 125 Phe Ile Glu Lys Val Leu Glu AsnAsn Tyr Thr Ala Leu Met Ser Ala 130 135 140 Lys Tyr Ser Gly Trp Tyr ValGly Phe Thr Lys Lys Gly Arg Pro Arg 145 150 155 160 Lys Gly Pro Lys ThrArg Glu Asn Gln Gln Asp Val His Phe Met Lys 165 170 175 Arg Tyr Pro LysGly Gln Pro Glu Leu Gln Lys Pro Phe Lys Tyr Thr 180 185 190 Thr Val ThrLys Arg Ser Arg Arg Ile Arg Pro Thr His Pro Ala 195 200 205 15 266 PRTHuman FGF-5 15 Ser Leu Ser Phe Leu Leu Leu Leu Phe Phe Ser His Leu IleLeu Ser 1 5 10 15 Ala Trp Ala His Gly Glu Lys Arg Leu Ala Pro Lys GlyGln Pro Gly 20 25 30 Pro Ala Ala Thr Asp Arg Asn Pro Arg Gly Ser Ser SerArg Gln Ser 35 40 45 Ser Ser Ser Ala Met Ser Ser Ser Ser Ala Ser Ser SerPro Ala Ala 50 55 60 Ser Leu Gly Ser Gln Gly Ser Gly Leu Glu Gln Ser SerPhe Gln Trp 65 70 75 80 Ser Leu Gly Ala Arg Thr Gly Ser Leu Tyr Cys ArgVal Gly Ile Gly 85 90 95 Phe His Leu Gln Ile Tyr Pro Asp Gly Lys Val AsnGly Ser His Glu 100 105 110 Ala Asn Met Leu Ser Val Leu Glu Ile Phe AlaVal Ser Gln Gly Ile 115 120 125 Val Gly Ile Arg Gly Val Phe Ser Asn LysPhe Leu Ala Met Ser Lys 130 135 140 Lys Gly Lys Leu His Ala Ser Ala LysPhe Thr Asp Asp Cys Lys Phe 145 150 155 160 Arg Glu Arg Phe Gln Glu AsnSer Tyr Asn Thr Tyr Ala Ser Ala Ile 165 170 175 His Arg Thr Glu Lys ThrGly Arg Glu Trp Tyr Val Ala Leu Asn Lys 180 185 190 Arg Gly Lys Ala LysArg Gly Cys Ser Pro Arg Val Lys Pro Gln His 195 200 205 Ile Ser Thr HisPhe Leu Pro Arg Phe Lys Gln Ser Glu Gln Pro Glu 210 215 220 Leu Ser PheThr Val Thr Val Pro Glu Lys Lys Asn Pro Pro Ser Pro 225 230 235 240 IleLys Ser Lys Ile Pro Leu Ser Ala Pro Arg Lys Asn Thr Asn Ser 245 250 255Val Lys Tyr Arg Leu Lys Phe Arg Phe Gly 260 265

That which is claimed is:
 1. A method for treating a human patient forcoronary artery disease, comprising administering into one or morecoronary vessels in a human patient in need of treatment for coronaryartery disease a therapeutically effective amount of an angiogenicallyactive fragment of a recombinant fibroblast growth factor (FGF) havingthe sequence set forth in SEQ ID NO: 3 or SEQ ID NO:
 5. 2. The method ofclaim 1, wherein said therapeutically effective amount administered tosaid patient is a unit dose of about 0.008 mg to about 6.1 mg of saidangiogenically active fragment of said recombinant FGF.
 3. The method ofclaim 2, wherein said therapeutically effective amount administered tosaid patient is a unit dose of 0.3 mg to 3.5 mg of said angiogenicallyactive fragment of said recombinant FGF.
 4. The method of claim 1,comprising administering into one or more coronary vessels of saidpatient about 0.2 μg/kg to about 36 μg/kg of said angiogenically activefragment of said recombinant FGF.
 5. The method of claim 4, comprisingadministering into one or more coronary vessels of said patient about0.2 μg/kg to about 2 μg/kg of said angiogenically active fragment ofsaid recombinant FGF.
 6. The method of claim 4, comprising administeringinto one or more coronary vessels of said patient about 2 μg/kg to about20 82 g/kg of said angiogenically active fragment of said recombinantFGF.
 7. The method of claim 4, comprising administering into one or morecoronary vessels of said patient about 20 μg/kg to about 36 μg/kg ofsaid angiogenically active fragment of said recombinant FGF.
 8. A methodfor treating a human patient for coronary artery disease, comprisingadministering into one or more coronary vessels in a human patient inneed of treatment for coronary artery disease a therapeuticallyeffective amount of an angiogenically active mutein of a recombinantfibroblast growth factor (FGF) having the sequence set forth in SEQ IDNO: 3 or SEQ ID NO:
 5. 9. The method of claim 8, wherein saidtherapeutically effective amount administered to said patient is a unitdose of about 0.008 mg to about 6.1 mg of said angiogenically activemutein of said recombinant FGF.
 10. The method of claim 9, wherein saidtherapeutically effective amount administered to said patient is a unitdose of 0.3 mg to 3.5 mg of said angiogenically active mutein of saidrecombinant FGF.
 11. The method of claim 8, comprising administeringinto one or more coronary vessels of said patient about 0.2 μg/kg toabout 36 μg/kg of said angiogenically active mutein of said recombinantFGF.
 12. The method of claim 11, comprising administering into one ormore coronary vessels of said patient about 0.2 μg/kg to about 2 μg/kgof said angiogenically active mutein of said recombinant FGF.
 13. Themethod of claim 11, comprising administering into one or more coronaryvessels of said patient about 2 μg/kg to about 20 μg/kg of saidangiogenically active mutein of said recombinant FGF.
 14. The method ofclaim 11, comprising administering into one or more coronary vessels ofsaid patient about 20 μg/kg to about 36 μg/kg of said angiogenicallyactive mutein of said recombinant FGF.
 15. A method for inducingangiogenesis in the heart of a patient, comprising administering intoone or more coronary vessels of a human patient in need of coronaryangiogenesis a therapeutically effective amount of a recombinantfibroblast growth factor (FGF) having the sequence set forth in SEQ IDNO: 3 or SEQ ID NO: 5 or an angiogenically active fragment or anangiogenically active mutein thereof.
 16. The method of claim 15,wherein said therapeutically effective amount administered to saidpatient is a unit dose of about 0.008 mg to about 6.1 mg of saidrecombinant FGF or said angiogenically active fragment or saidangiogenically active mutein thereof.
 17. The method of claim 15,wherein said unit dose is administered into two coronary vessels of saidpatient.
 18. The method of claim 15, further comprising the step ofadministering to said patient an effective amount of a glycosaminoglycanwithin 30 minutes of administering said recombinant FGF or saidangiogenically active fragment or said angiogenically active muteinthereof.
 19. A unit dose of a fibroblast growth factor (FGF), comprisingabout 0.008 mg to about 6.1 mg of an FGF having the amino acid sequenceof SEQ ID NO: 3 or SEQ ID NO: 5 or an angiogenically active fragment oran angiogenically active mutein thereof.
 20. The unit dose of claim 19,comprising 0.3 mg to 3.5 mg of said FGF or said angiogenically activefragment or said angiogenically active mutein thereof.
 21. The unit doseof claim 19, wherein said FGF has the sequence of SEQ ID NO: 3 or SEQ IDNO:
 5. 22. A pharmaceutical composition comprising (i) a therapeuticallyeffective amount of a fibroblast growth factor (FGF) having the sequenceof SEQ ID NO: 3 or SEQ ID NO: 5 or an angiogenically active fragment oran angiogenically active mutein thereof, and (ii) a pharmaceuticallyacceptable carrier, said composition being in a form and a size suitablefor administration to a human patient.
 23. The pharmaceuticalcomposition of claim 22, wherein said therapeutically effective amountcomprises about 0.2 μg/kg to about 36 μg/kg of said FGF or saidangiogenically active fragment or said angiogenically active muteinthereof.
 24. The pharmaceutical composition of claim 23, wherein saidtherapeutically effective amount comprises about 0.2 μg/kg to about 2μg/kg of said FGF or said angiogenically active fragment or saidangiogenically active mutein thereof.
 25. The pharmaceutical compositionof claim 23, wherein said therapeutically effective amount comprisesabout 2 μg/kg to about 20 μg/kg of said FGF or said angiogenicallyactive fragment or said angiogenically active mutein thereof.
 26. Thepharmaceutical composition of claim 23, wherein said therapeuticallyeffective amount comprises about 20 μg/kg to about 36 μg/kg.
 27. Thepharmaceutical composition of claim 22, wherein said therapeuticallyeffective amount comprises about 008 mg to about 6.1 mg of said FGF orsaid angiogenically active fragment or said angiogenically active muteinthereof.
 28. The pharmaceutical composition of claim 27, wherein saidtherapeutically effective amount comprises 0.3 mg to 3.5 mg of said FGFor said angiogenically active fragment or said angiogenically activemutein thereof.